WO2011014872A2

WO2011014872A2 - Compositions and methods for diagnosing, treating or preventing neoplasias

Info

Publication number: WO2011014872A2
Application number: PCT/US2010/044118
Authority: WO
Inventors: Anirban Maitra; Hector Alvarez
Original assignee: The Johns Hopkins University
Priority date: 2009-07-31
Filing date: 2010-08-02
Publication date: 2011-02-03
Also published as: WO2011014872A3

Abstract

As described below, the present invention features compositions and methods for diagnosing, treating and preventing neoplasias.

Description

COMPOSITIONS AND METHODS FOR DIAGNOSING, TREATING OR

PREVENTING NEOPLASIAS

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of the following U.S. Provisional Application No.:

61/230, 157, filed July 31 , 2009, and 61/234,808, filed August 18, 2009, the entire contents of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

This work was supported by the following grants from the National Institutes of Health, Grant Nos: P50CA062924. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The incidence of esophageal adenocarcinoma (EAC) is increasing at an alarming pace in the United States (>600% increase since 1975). Established epidemiological risk factors for EAC include male gender, Caucasian ethnicity, abdominal obesity, pre-existing gastroesophageal reflux disease (GERD), and most importantly, Barrett esophagus, a metaplastic precursor lesion to EAC. In the multistep carcinogenesis model of EAC, metaplastic Barrett epithelium progresses through the intermediate stages of low-grade and high-grade dysplasia, culminating in invasive cancer. Unfortunately, the overwhelming majority of patients (>90%) with EAC present de novo, i.e. without a pre-existing diagnosis of Barrett esophagus. Patients with de novo EAC are almost always diagnosed at an advanced stage of disease, which likely accounts for their dismal overall five-year survival (- 13%). The treatment of choice for advanced esophageal cancer is esophagectomy, usually preceded by neo-adjuvant chemo- radiation therapy. However, despite the advances in treatment, and an increase in the proportion of cases diagnosed at an early, and hence potentially curable stage, the overall five-year survival of EAC has changed only minimally in the last 3 decades, underscoring the dire need for developing more potent therapies for this malignancy.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions and methods for diagnosing, treating and preventing neoplasias. In one aspect, the invention generally provides therapeutic methods featuring

compositions, including pharmaceutical composition for the treatment of a neoplasia (e.g., a cancer of epithelial, mesenchymal, hematological, or neural/neuroectodermal origin) containing^, an effective amount of an agent that inhibits the expression or activity of AxI in combination with an effective amount of an agent that inhibits the expression or activity of EGF receptor and/or Her2. In one embodiment, the agent that inhibits the expression or activity of AxI is a small compound (e.g. R428 or a derivative or structurally related compound thereof), an anti-Axl antibody, or an AxI inhibitory nucleic acid molecule (e.g., antisense, siRNA, shRNA). In another embodiment, the agent that inhibits the expression or activity of EGF receptor is a small compound, an anti-EGF receptor antibody, or an EGF receptor inhibitory nucleic acid molecule (e.g., antisense, siRNA, shRNA). In another embodiment, the agent is gefitinib (Iressa), Cetuximab (Erbitux), Erlotinib (Tarceva), Lapatinib, Panitumumab (Vectibix, ABX-EGF), RO5083945, IMC-11F8 (necitumumab), BMS-690514, BIBW2992 (Tovok), PF-00299804, CUDC-101, or BIOMAb-EGFR (Nimotuzumab). In one embodiment, the agent that inhibits the expression or activity of a Her2 receptor is a small compound, an anti-Her2 receptor antibody, or an Her2 inhibitory nucleic acid molecule (e.g., antisense, siRNA, shRNA). In another embodiment, the agent is Trastuzumab (Herceptin^®), Lapatinib, or BIBW2992 (Tovok). In other embodiments, the neoplasia is lung cancer, esophogeal carcinoma, colorectal carcinoma, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, endometrial cancer, melanoma, glioblastoma, astrocytoma, or acute leukemia.

In another aspect, the invention provides methods for treating or preventing a neoplasia, the method involving administering to a subject an effective amount of an agent that inhibits the expression or activity of AxI in combination with an agent that inhibits the expression or activity of EGF receptor and/or Her2.

In another aspect, the invention provides methods of reducing the invasiveness of an esophageal adenocarcinoma cell in a subject, the method comprising contacting the neoplastic cell with an agent that inhibits AXL expression or biological activity in the cell relative to an untreated control cell.

In another aspect, the invention provides methods of treating or preventing esophageal adenocarcinoma in a subject, the method comprising administering to the subject an agent that inhibits AXL expression or biological activity in the subject relative to a reference. In still another aspect, the invention provides methods for inhibiting the progression of Barrett esophagus to esophageal adenocarcinoma in a subject, the method comprising

administering to the subject an agent that inhibits inhibits AXL expression or biological activity in the cell relative to an untreated control cell.

In yet another aspect, the invention provides methods for inhibiting the invasiveness of an esophageal adenocarcinoma in a subject, the method comprising administering to the subject an agent that inhibits AXL expression or biological activity in the cell relative to an untreated control cell.

In yet another aspect, the invention provides pharmaceutical composition containing an effective amount of one or more agents sufficient to inhibit the progression of Barrett esophagus to esophageal adenocarcinoma in a subject, and a pharmaceutically-acceptable carrier.

In yet another aspect, the invention provides pharmaceutical composition comprising an effective amount of one or more agents sufficient to treat, prevent, or reduce the invasiveness of esophageal adenocarcinoma in a subject, and a pharmaceutically-acceptable carrier. In various embodiments of the above aspect, the agent is R428. In other embodiments, the method of composition further comprises administering an effective amount of lapatinib or an EGF receptor and/or Her2 receptor antagonist.

In yet another aspect, the invention provides a kit for the treatment of a neoplasia, the kit containing the pharmaceutical composition of any previous aspect or any other aspect of the invention delineated herein and directions for using the kit for the treatment of a esophageal adenocarcinoma or for the prevention of the progression of Barrett esophagus to esophageal adenocarcinoma.

In yet another aspect, the invention provides a method for characterizing Barrett esophagus in a subject, the method comprising comparing the expression or activity of an AxL polypeptide or polynucleotide in a biological sample of the subject to a reference level. In one embodiment, the reference is the level of AxL present in a healthy control sample, a esophageal adenocarcinoma sample, or a sample from the same subject at an earlier point in time. In another embodiment, the sample is a sample of esophageal epithelium or a lymph node sample. In yet another embodiment, failure to detect or detection of minimal AxI expression indicates the presence of a non-dysplastic Barrett epithelium or a low-grade dysplasia. In another

embodiment, increased AxI expression indicates the presence of a high-grade dysplasia. In another embodiment, an increased level of AxL in Barrett esophagus relative to a sample obtained at an earlier time point from the same subject indicates disease progression. In another embodiment, AxL expression is measured in an immunoassay, a radioassay, or other standard method.

In yet another aspect, the invention provides a method for diagnosing a subject as having or having a propensity to develop esophageal adenocarcinoma, the method involving detecting AxL expression or biological activity in a biological sample from the subject relative to a healthy control, thereby diagnosing esophageal adenocarcinoma in the subject.

In yet another aspect, the invention provides a method for selecting an appropriate treatment for a subject having Barrett esophagus or esophageal adenocarcinoma, the method comprising measuring the expression or activity of AxL in a sample from the subject, wherein the level of AxL expression or activity indicates an appropriate treatment. In one embodiment, minimal to largely absent AxI expression indicates that the subject should be monitored for progression of Barrett esophagus to esophageal adenocarcinoma. In another embodiment, the subject is identified as in need of preventive therapy with an agent that inhibits the expression or activity of AxI. In one embodiment, an increased level of AxI expression in an esophageal biopsy indicates that the subject is in need of aggressive treatment including multimodality chemotherapy with or without radiation to the cancer field. In another embodiment, the subject is identified as in need of treatment with an agent that inhibits the expression or activity of AxI. In another embodiment, the agent is R428. In another embodiment, the treatment further comprises administering lapatinib or another EGF receptor antagonist to the subject. In another embodiment, the sample comprises lymph node tissue. In another embodiment, the

identification of AxI expression or activity in lymph node tissue indicates that the subject has metastatic esophageal adenocarcinoma.

In yet another aspect, the invention provides a method for monitoring the condition of a subject having Barrett's esophagus, the method comprising comparing the level of AxI expression or activity in a biological sample from the subject with the level present in a sample obtained at an earlier time, wherein a reduction in said AxI level identifies an improvement in the subject's condition, and an increase in said AxI level identifies a worsening in the subject's condition. In yet another aspect, the invention provides a method for determining the prognosis of a subject having Barrett's esophagus or esophageal adenocarcinoma, the method comprising the level of AxI expression or activity in a biological sample from the subject with the level present in a reference, wherein the level of AxI in the sample is indicative of the subject's prognosis. In one embodiment, an increased level of AxI indicates a poor prognosis and an increased level indicates a better prognosis.

In yet another aspect, the invention provides a therapeutic composition for the cell- specific targeting of a therapeutic agent to an Axl-expressing cell, the composition containing an anti-Axl antibody, aptamer, or other AxI binding agent linked to a chemotherapeutic agents, toxin or active fragments thereof, or radioactive isotope.

In yet another aspect, the invention provides a diagnostic composition for the cell- specific targeting of a detectable agent to an Axl-expressing cell, the composition comprising an anti-Axl antibody, aptamer, or other AxI binding agent linked to a detectable moiety.

In various embodiments of the above aspect or any other aspect of the invention delineated herein, the agent that inhibits the expression or activity of AxI is a small compound AxI inhibitor (e.g., R428 and analogs and derivatives thereof), an anti-Axl antibody, or an AxI inhibitory nucleic acid molecule. In other embodiments, the agent that inhibits the expression or activity of EGF receptor is a small compound, an anti-EGF receptor antibody, or an EGF receptor inhibitory nucleic acid molecule. In other embodiments, the agent that inhibits the expression or activity of a Her2 receptor is a small compound, an anti-Her2 receptor antibody, or an Her2 inhibitory nucleic acid molecule. In certain preferred embodiments of the methods of the invention, the agent that inhibits the expression or activity of AxI is administered alone or in combination with an agent that inhibits the expression or activity of EGF receptor or an agent that inhibits the expression or activity of a Her2 receptor. In various embodiments of the above aspects, the neoplasia is a cancer of epithelial, mesenchymal, hematological, or

neural/neuroectodermal origin. In other embodiments, the agent that inhibits the expression or activity of AxI is a small compound (e.g. R428 or a derivative or structurally related compound thereof), an anti-Axl antibody, or an AxI inhibitory nucleic acid molecule (e.g., antisense, siRNA, shRNA). In other embodiments, the agent that inhibits the expression or activity of EGF receptor is a small compound, an anti-EGF receptor antibody, or an EGF receptor inhibitory nucleic acid molecule (e.g., antisense, siRNA, shRNA). In other embodiments, the agent is gefitinib (Iressa), Cetuximab (Erbitux), Erlotinib (Tarceva), Lapatinib, Panitumumab (Vectibix, ABX-EGF), RO5083945, IMC-1 1F8 (necitumumab), BMS-690514, BIBW2992 (Tovok), PF- 00299804, CUDC-101 , or BIOMAb-EGFR (Nimotuzumab). In one embodiment, the agent that inhibits the expression or activity of a Her2 receptor is a small compound, an anti-Her2 receptor antibody, or an Her2 inhibitory nucleic acid molecule (e.g., antisense, siRNA, shRNA). In another embodiment, the agent is Trastuzumab (Herceptin^®), Lapatinib, or BIBW2992 (Tovok). In other embodiments, the neoplasia is lung cancer, esophogeal carcinoma, colorectal carcinoma, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, endometrial cancer, melanoma, glioblastoma, astrocytoma, or acute leukemia. In other embodiments, the cell is in vitro or in vj^'vo. In other embodiments, the agent is an AXL inhibitory nucleic acid molecule that is an antisense polynucleotide, siRNA or an shRNA molecule. In other embodiments, the agent is a small compound that inhibits AxL receptor tyrosine kinase activity. In other embodiments, the " agent is R428. In other embodiments, the agent is an AXL -specific antibody. In other embodiments, the method further comprises administering to the subject an effective amount of lapatinib and/or Iressa. In other embodiments, the subject is a human. In other embodiments, the agent reduces neoplastic cell proliferation or invasiveness relative to the level in a corresponding neoplastic cell.

The invention provides compositions and methods for diagnosing and treating esophageal neoplasias. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

By "reducing the invasiveness" is meant reducing the propensity of a neoplasia to metastasize. In particular embodiments, an agent that reduces the invasiveness of a neoplastic cell inhibits the migration, engraftment or invasion of that cell into other tissues or organs. The invasiveness of a neoplastic cell can be measured in vitro or in vivo.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. "

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. Accordingly, the invention provides analogs, derivatives, and compounds that are structurally related to any one of the following: R428, gefitinib (Iressa), Trastuzumab (Herceptin^®), Cetuximab (Erbitux), Erlotinib (Tarceva), Lapatinib, Panitumumab (Vectibix, ABX-EGF), as well as emerging therapies against these receptors currently undergoing evaluation, such as RO5083945, IMC-11F8 (necitumumab), BMS-690514, BIBW2992 (Tovok), PF-00299804, CUDC-101 , and BIOMAb-EGFR (Nimotuzumab).

"Biological sample" as used herein refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid origin, obtained, reached, or collected in vivo or in situ, that contains or is suspected of containing nucleic acids or polypeptides of AxI. A biological sample also includes samples from a region of a biological subject containing precancerous or cancer cells or tissues. Such samples can be, but are not limited to, organs, tissues, fractions and cells isolated from mammals including, humans such as a patient, mice, and rats. Biological samples also may include sections of the biological sample including tissues, for example, frozen sections taken for histologic purposes.

A "cancer" in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. In some circumstances, cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells, for example, leukemic cells.

By "chemotherapeutic agent" is meant an agent that is used to kill cancer cells or to slow their growth. Accordingly, both cytotoxic and cytostatic agents are considered to be

chemotherapeutic agents. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

The phrase "detecting a neoplasia" or "diagnosing a neoplasia" refers to determining the presence or absence of cancer or a precancerous condition in an animal. "Detecting a cancer" also can refer to obtaining indirect evidence regarding the likelihood of the presence of precancerous or cancerous cells in the animal or assessing the predisposition of a patient to the development of a cancer. Detecting a cancer can be accomplished using the methods of this invention alone, in combination with other methods, or in light of other information regarding the state of health of the animal.

By "detectable moiety" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include Barrett esophagus, esophageal adenocarcinoma, and other neoplasias, including those of epithelial, mesenchymal, hematological, or neural/neuroectodermal origin.

By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

The phrase "having a propensity to develop" refers to the probability or risk that a subject will develop a particular pathological condition.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

By "immunological assay" is meant an assay that relies on an immunological reaction, for example, antibody binding to an antigen. Examples of immunological assays include ELISAs, Western blots, immunoprecipitations, and other assays known to the skilled artisan.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "neoplasia" is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Examples of cancers include, without limitation, esophageal adenocarcinoma, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocyte leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease),

Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,

lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate 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, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,

hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

The term "precancerous" refers to cells or tissues having characteristics relating to changes that may lead to malignancy or cancer. A tissue affected by Barrett esophagus may, in some circumstances, be considered precancerous.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By "reference" is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison. The sequences of an exemplary AxI polypeptide and AxI polynucleotide are reference sequences that are provided herein below. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol.

152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1 % SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1 % SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961 , 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence.

By "siRNA" is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21 , 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.

By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

A "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, or 0.01 % of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures IA and IB show a Serial Analysis of Gene Expression (SAGE) in Barrett esophagus progression model. Figure IA provides a schematic diagram illustrating endoscopic mucosal biopsies obtained from a single individual who progressed to EAC during surveillance. The corresponding histological diagnoses are indicated, based on light microscopic examination of cryostat embedded sections. Figure IB is a schematic diagram and a Heat map illustrating a subset of the most highly differentially expressed transcripts in the multistep progression of Barrett esophagus. L-SAGE tag corresponding to the AXL gene is differentially upregulated during the progression to EAC.

Figures 2A-2C show that AxI overexpression is an adverse prognostic factor in EAC. Figure 2A provides an immunohistochemical analysis of AxI expression performed in multistage Barrett progression using a panel of archival tissue microarrays. AxI expression restricted to the basal (regenerative) layer of normal squamous epithelium was observed in a minor subset of cases). Minimal to largely absent AxI expression was observed in non-dysplastic Barrett epithelium and in low-grade dysplasia. Robust AxI expression was observed in approximately a quarter of high-grade dysplasia. AxI was overexpressed in -60% of primary EACs, and in a comparable percentage of lymph node metastases. Figure 2B is a histogram that summarizes the frequencies of tissue "cores" in each histopathologic category with high levels of AxI expression. Figure 2C shows a Kaplan Meier survival analysis of 92 Barrett-associated EAC demonstrates that cancers with high AXL expression have a significantly worst median survival than cancers with absent/low AXL expression (Log rank test P = 0.036).

Figures 3A-3D show that immunohistochemical analysis confirms upregulation of the

AxI ligand, Gas6, in primary and metastatic EAC. Figure 3A includes micrographs showing GAS6 overexpression during the entire histological spectrum of Barrett progression is shown for one single patient, demonstrating upregulation in the core representing EAC and metastasis. Figure 3B is a histogram that summarizes the frequencies of tissue "cores" in each

histopathologic category with high levels of Gas6 expression. Upregulation of ligand expression was predominantly observed at the stage of EAC and metastases. These analyses were performed on the identical tissue microarrays used in Figure 2. Figure 3C is a Kaplan Meier survival analysis of 92 Barrett-associated EACs, which demonstrates no statistically significant differences in median survival for cancers with absent/low GAS6 versus high expression. Figure 3D is a graph showing the 92 cases are sub-classified into four cohorts, based on relative levels of AxI and Gas6 expression. The expression levels of the AxI receptor, rather than Gas6 ligand, appear to be the limiting factor and determinant of prognosis.

Figures 4A-4C show the overexpression of AxI in EAC cell lines, and efficient knockdown of endogenous AxI protein using lentiviral short hairpin RNA (shRNA). Figure 4A is a Western blot showing that AxI was overexpressed in EAC cells compared to non-neoplastic esophageal keratinocyte lines. Western blot analysis demonstrated upregulation of AxI protein in JH-EsoAdl and OE33 EAC cell lines compared to NEK2 and HEEPIC human esophageal keratinocyte lines. The level of overexpression is particularly striking for JH-EsoAdl cells. Detectable Gas6 protein was only observed in JH-EsoAdl and NEK2 cells. HeLa cells were used as positive control, and actin as housekeeping control. Figure 4B is a histogram showing that endogenous AxI was downregulated in the JH-EsoAdl and OE33 cells using a lentiviral shRNA vector (Open Biosystems). Compared to scrambled shRNA infected ("mock") clones, the shRNA-expressing cells demonstrated a significant loss of AXL transcripts {left) and a complete loss of the AxI protein expression.

Figure 5 illustrates the genomic amplification of the AXL locus in JH-EsoAdl cells. {Top) Array CGH (244K Agilent microarrays) identified a chromosome 19ql2-13.1

amplification, which contains the AXL gene locus, in JH-EsoAdl cells. {Bottom, left)

Quantitative genomic PCR for three AXL exons (represented by white, gray, and black bars, respectively) in JH-EsoAdl cells identified increased gene dosage compared to the matched DNA from EBV-transformed lymphoblastoid cells (i.e., germline DNA), confirming the somatic nature of the amplification. DOCK6, a gene located within a copy number neural region of chromosome 19p, was used as housekeeping control. {Bottom, right) FISH analysis in JH- EsoAdl cells using a chromosome 19q 13.1 BAC spanning the AXL gene confirms the increased copy number.

Figures 6A-6F show that AxI regulates multiple facets of the transformed phenotype in EAC cell lines. Figures 6A and 6B are graphs showing that the loss of endogenous AxI function significantly inhibited migration at 24 hours in JH-EsoAdl and OE33 cells, as assessed by a modified Boyden chamber assay. Error bars = +S.E.M. (N = 3); P < 0.05, compared with scrambled. Figures 6C and 6D are graphs showing that the loss of endogenous AxI function significantly inhibits invasion at 48 hours in JH-EsoAdl and OE33 cells, as assessed by a modified Boyden chamber assay. Error bars = ±S.E.M. (N = 3); P < 0.05, compared with scrambled. Figure 6E shows that the loss of endogenous AxI function significantly reduced the property of anchorage independent growth (assessed by colony formation in soft agar) in OE33 cells. Higher magnification of soft agar assay is shown on top. Error bars = ±S.E.M. (N = 3); P < 0.05, compared with scrambled. Figure 6F shows that loss of endogenous AxI function abrogated the ability of JH-EsoAdl cells to engraft in NOD/SCDD mice. In three animals, xenografts were visible in all flanks implanted with scrambled infected JH-EsoAdl cells (white arrow), while no xenografts formed on the opposite flank, corresponding to cells expressing AXL shRNA. Histological analysis of explanted scrambled expressing JH-EsoAdl xenograft confirms adenocarcinoma histology {bottom, right).

Figures 7A-7F show that sustained AxI function is required for maintaining EGF- dependent activation of RaI GTPase proteins. Figure 6A is a Western blot showing that Gas6 ligand induced tyrosine phosphorylation of the AxI receptor in JH-EsoAdl cells, which is accompanied by phosphorylation of Akt at Ser⁴⁷³, and p42/p44 MAPK (Lanes 1 and 3).

Knockdown of endogenous AxI completely abrogated Gas6-induced Akt phosphorylation, and modestly inhibited Gas6-induced p42/p44 MAPK phosphorylation (Lane 4). Phosphorylation of the AxI receptor was assessed by immunoprecipitation with PY99 anti-phosphotyrosine antibody, followed by Western blot with anti-Axl antibody. The Gas6 ligand induced tyrosine phosphorylation of AxI receptor in OE33 cells, which was accompanied by phosphorylation of Akt at Ser⁴⁷³, and p42/p44 MAPK (Lanes 1 and 3). In contrast to JH-EsoAdl cells, knockdown of endogenous AxI had minimal effects on Gas6-induced phosphorylation of either Akt or p42/p44 MAPK. Figure 7C shows that in both JH-EsoAdl and in OE33 cell lines, knockdown of AxI profoundly decreased EGF-dependent activation of RaI GTPase isoforms (RaIA and RaIB). Activation of RaI proteins was assessed by immunoprecipitation for GTP-bound moieties in scrambled and AXL shRNA expressing clones, followed by Western blot analysis for the respective RaI isoform. Figure 7D shows that in both JH-EsoAdl and in OE33 cell lines, loss of RaI GTPase activity was also accompanied by decreased levels of GTP-bound (active) Cdc42, a Rho GTPase family member that is a credentialed RaI effector protein. Figure 7E shows that expression of a constitutively active form (RIf-CAAX) of the RaIGEF, Rgl2, in OE33 cells with AxI knockdown restituted the levels of GTP-bound RaIA protein to that observed in control cells in the presence of EGF ligand. Lane 1: Mock vector expressing OE33 cells with stable RIf- CAAX expression; Lane 2: Scrambled expressing OE33 cells; Lane 3: OE cells with stable AXL shRNA expression; Lane 4: OE cells with stable co-expression of AXL shRNA and RIf-CAAX. Figure 7F shows that restitution of RaIA activity is associated with partial, but significant, rescue of cell motility in OE33 cells, including migration (left) and invasion (right) phenotypes. * = significant downregulation of cell motility in AXL shRNA expressing cells compared to scrambled (P<0.05); ** = significant rescue of cell motility in AXL shRNA and RIf-CAAX co- expressing cells compared to cells with AxI knockdown alone.

Figures 8A-8H AxI regulates tyrosine phosphorylation of the EGFR and ERBB2 receptors, and inhibition of AxI function modulates sensitivity to EGFR and EGFR/ERBB2 dual kinase antagonists. Figure 8A is a Western blot showing that stimulation of serum-starved OE33 cells with rhGasό ligand (200ng/mL) lead to increased tyrosine phosphorylation of the AxI receptors, as well as EGFR and ERBB2 at 15 minutes. Protein lysates were obtained at 5, 15, and 30 minutes post-Gasό stimulation, immunoprecipitated with anti-phosphotyrosine (PY99) antibody, and Western blot performed for AxI, EGFR, and ERBB2. Figure 8B is a Western blot showing that stimulation of serum-starved OE33 cells with rhGasό ligand (200ng/mL) lead to increased tyrosine phosphorylation of the AxI receptors, as well as EGFR and ERBB2 at 15 minutes. Protein lysates were obtained at 5, 15, and 30 minutes post-Gasό stimulation, immunoprecipitated with anti-Axl antibody, and Western blot performed for phosphotyrosine (with PY99), EGFR, and ERBB2. Of note, both EGFR and ERBB2 were present in the AxI "pull down" even in the absence of Gasό, but the protein-protein interaction increased upon ligand stimulation, coinciding with the time point of maximal tyrosine phosphorylation. Figures 8C and 8D are Western blots showing that stimulation of serum-starved OE33 cells with rhGasό ligand (200ng/mL) lead to increased tyrosine phosphorylation of Tyr⁸⁷⁷ residue of ERBB2 at 15 minutes. Phosphorylation at this residue was essentially abrogated in OE33 cells with AXL shRNA knockdown, either in absence or presence of Gasό ligand. Thus, sustained AxI function waaas required for the maintenance of Tyr⁸⁷⁷ phosphorylation of ERBB2 in OE33 cells. Figure 8E is a graph showing that the AxI small molecule inhibitor R428 significantly downregulated in vitro invasion (modified Boyden chamber assay) at 48 hours in OE33 cells (left) and significantly blocked colony formation in soft agar at two weeks (right) compared to vehicle treated cells. Two doses (2μM and 4μM) were used in these experiments. Figure 8F is a Western blot showing that R428 inhibited Axl-dependent tyrosine phosphorylation of the ERBB2 Tyr⁸⁷⁷ residue (second row), as well as the protein-protein interaction between AxI and ERBB2 (third row), and between AxI and EGFR (fourth row), in a dose-dependent manner in OE33 cell line. Total AxI protein levels were unaffected by R428; GAPDH was used as loading control. Figure 8G is a graph showing that MTS assays in OE33 cells confirmed that addition of R428 sensitized this cell line to the EGFR tyrosine kinase inhibitor gefitinib, and the

EGFR/ERBB2 dual tyrosine kinase inhibitor lapatinib. A reduction in IC₅₀ was observed for gefitinib (lOμM to 0.9μM) and lapatinib (5.5μM to 0.4μM), when co-treated with R428 versus the single agent. Figure 8H is a graph showing that OE33 cells, stably expressing either scrambled or AXL shRNA lentivirus, were treated with gefintinib and lapatinib, and decreased IC₅₀ was observed for both small molecules with loss of AxI function (from 8.7μM to 3.2μM for gefitinib, and 4.8μM to 1.8μM for lapatinib, respectively). Points in both graphs represent the mean+SEM of three replicates from individual experiments. IC₅₀ values are given on each graph's table.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods that are useful for diagnosing, treating and preventing neoplasia.

The invention is based, at least in part, on the discovery that the AxI receptor tyrosine kinase (AxI) was significantly and progressively upregulated during multistep esophageal carcinogenesis, and that reducing the expression or activity of AxI reversed key aspects of the neoplastic phenotype, including inhibiting invasion, migration, and engraftment. Moreover, levels of AxI were identified as an independent adverse prognostic factor in subjects having esophageal adenocarcinoma resection. Significantly, it was also discovered that agents that reduce the expression or activity of AxI sensitized neoplastic cells to EGF receptor and ERBB2 (Her-2/neu) receptor antagonists. Accordingly, AxI is particularly useful for reducing the levels of chemotherapeutics (e.g., EGF receptor or ERBB2 receptor antagonists) required to for the treatment of a neoplasia and for the treatment of refractory neoplasias that are not susceptible to treatment with conventional chemotherapeutic agents. Accordingly, the invention provides therapeutic methods, including therapeutic compositions comprising an AxI inhibitor in combination with an EGF receptor or ERBB2 receptor antagonist, and methods of using such compositions for the treatment of neoplasias. In other embodiments, the invention provides methods for treating or preventing the progression of Barrett esophagus to esophageal adenocarcinoma, methods for determining the prognosis and/or probability of survival of subjects identified as having Barrett esophagus or esophageal adenocarcinoma by determining the level of AxI polypeptide or polynucleotide in a biological sample from the subject, methods for selecting appropriate therapeutic regimens for subjects that have or have a propensity to develop esophageal adenocarcinoma, and methods for diagnosing and imaging Axl-expressing cells in subjects that have or have a propensity to develop Barrett esophagus or esophageal adenocarcinoma.

EGF Receptors and Cancer

The family of epidermal growth factor receptors (EGFR; HERl, HER2/neu, HER3, and HER4) are cell membrane receptors having tyrosine kinase activity, which play a key role in the behavior of malignant cells in a variety of human cancers (e.g., lung cancer, esophogeal carcinoma, colorectal carcinoma, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, endometrial cancer, melanoma, glioblastoma, astrocytoma, or acute leukemia, and other solid cancers ). Presently, EGF receptor family antagonists, including anti-HER2 monoclonal antibodies, anti-EGF receptor MAbs and EGF receptor and HER2 tyrosine kinase inhibitors, are used in combination with conventional chemotherapeutics. While these combination therapies increase survival of some patients, many patients ultimately succumb to metastatic disease. There are patients whose cancers are susceptible to treatment with existing therapies, nevertheless, many of them suffer from adverse side-effects due to the high dosages of toxic chemotherapeutics that they receive.

The present invention identifies a new combination therapy that targets AxI in combination with the EGF receptor and/or ERBB2/HER2. The use of an agent that inhibits the expression or activity of AXL sensitizes the neoplastic cells to treatment with EGF receptor and/or ERBB2/HER2 antagonists, thereby providing imporved methods for the treatment of cancers of epithelial, mesenchymal, hematological, or neural/neuroectodermal origin, particularly those that are resistant to conventional therapies. Advantageously, the present invention permits the use of lower dosages of concurrently administered chemotherapeutics, thereby reducing adverse side effects.

Therapy

The invention provides agents that reduce the expression or activity of AxI. Such agents include small molecule inhibitors of AxI tyrosine kinase activity, such as R428, anti-Axl antibodies, and inhibitory nucleic acid molecules that target AxI (e.g., antisense nucleic acid molecule, siRNAs, shRNAs). Such agents may be used alone or in combination with agents that inhibit the expression or activity of the EGF receptor and/or ErBB2/Her2. Therapy comprising agents of the invention may be provided wherever cancer therapy is performed: at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment generally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed. The duration of the therapy depends on the kind of cancer being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient's body responds to the treatment. Drug administration may be performed at different intervals (e.g., daily, weekly, or monthly). Therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to build healthy new cells and regain its strength. Depending on the type of cancer and its stage of development, the therapy can be used to slow the spreading of the cancer, to slow the cancer's growth, to kill or arrest cancer cells that may have spread to other parts of the body from the original tumor, to relieve symptoms caused by the cancer, or to prevent cancer in the first place. As used herein, the terms "cancer" or "neoplasm" or "neoplastic cells" is meant a collection of cells multiplying in an abnormal manner. Cancer growth is uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells.

The present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to neoplasms (e.g., lung cancer, esophogeal carcinoma, colorectal carcinoma, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, endometrial cancer, melanoma, glioblastoma, astrocytoma, or acute leukemia). In particular embodiments, the invention provides a method of treating Barrett esophagus, esophageal adenocarcinoma, or other neoplastic diseases or disorders associated with increased AxI expression or activity or symptoms thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to inhibit the expression or biological activity of AxI, thereby treating the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein (e.g., R428 alone or in combination with, for example, anti-HER2 monoclonal antibodies, anti-EGF receptor MAbs and/or EGF receptor or HER2 receptor tyrosine kinase inhibitors), or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which increased expression or activity of AxI, EGFR, and/or ErBB2/Her2 may be implicated. In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., AxI or any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with increased AxI expression or activity, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

Compounds of the Invention

The AxI inhibitor R428, as well as inhibitory nucleic acid molecules targeting AxI, inhibited the migration, invasion, and anchorage independent growth of such cells. These agents also sensitized the cells to treatment with lapatinib and gefitinib (Iressa). Accordingly, the invention provides methods for treating neoplasias by administering an AxI antagonist alone, or in combination with an EGF receptor and or HER2 receptor antagonist. Interestingly, R428 was found to inhibit the transformed phenotype of esophageal adenocarcinoma cells, and was particularly effective when administered in combination with lapatinib. These compounds, and others having AxI, EGFR, or Her2 receptor inhibitory activity, are useful in combination for the treatment of neoplaisa. In particular embodiments, for preventing, slowing, or otherwise inhibiting the progression of Barrett esophagus to esophageal adenocarcinoma.

Combination Therapies

Agents of the invention include small compounds that reduce the expression or activity of

AxI, anti-Axl antibodies, and inhibitory nucleic acid molecules that target AxI. As described herein below, such agents are particularly useful for sensitizing cancers of epithelial, mesenchymal, hematological, or neural/neuroectodermal origin to an anti-neoplasia therapeutic, such as an agent that inhibits the expression or activity of an EGF receptor and/or ErBB2/Her2. Such agents include, but are not limited to anti-HER2 monoclonal antibodies, small compounds that inhibit HER2, Her2 inhibitory nucleic acid molecules, anti-EGF receptor monoclonal antibodies, EGF receptor tyrosine kinase inhibitors, and EGF receptor inhibitory nucleic acid molecules. In particular embodiments, an agent that inhibits AxI expression or activity (e.g., R428) is administered in combination with one or more of the following: gefitinib (Iressa), Trastuzumab (Herceptin^®), Cetuximab (Erbitux), Erlotinib (Tarceva), Lapatinib, Panitumumab (Vectibix, ABX-EGF), as well as emerging therapies against these receptors currently undergoing evaluation, such as RO5083945, DVIC-1 1F8 (necitumumab), BMS-690514,

BIBW2992 (Tovok), PF-00299804, CUDC-101 , and BIOMAb-EGFR (Nimotuzumab) Such agents may be administered in combination with any other standard anti-neoplasia therapy; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin.

Barrett Esophagus and Esophageal Adenocarcinoma

Esophageal adenocarcinoma (EAC) is associated with a reflux-induced metaplastic phenomenon known as Barrett esophagus. Serial Analysis of Gene Expression (SAGE) was performed on metachronous mucosal biopsies from a patient who underwent progression to esophageal adenocarcinoma during endoscopic surveillance. SAGE confirmed significant upregulation of AxI "tags" during the multistep progression of Barrett esophagus to esophageal adenocarcinoma. In a cohort of 92 surgically resected esophageal adenocarcinomas, AxI overexpression was associated with shortened median survival on both univariate (P<0.004) and multivariate (f<0.036) analysis. Genetic knockdown of AxI receptor tyrosine kinase (RTK) function was enabled in two esophageal adenocarcinoma cell lines (OE33 and JH-EsoAdl) using lentiviral short hairpin RNA (shRNA). Genetic knockdown of AxI in EAC cell lines inhibited invasion, migration, and in vivo engraftment, which was accompanied by downregulation in the activity of the RaI GTPase proteins (RaIA and RaIB). Restoration of RaI activation rescued the transformed phenotype of esophageal adenocarcinoma cell lines, suggesting a novel effector mechanism for AxI in cancer cells. Pharmacological inhibition of AxI was carried out using a small molecule antagonist, R428 (Rigel Pharmaceuticals). Pharmacological inhibition of AxI with R428 in EAC cell lines significantly reduced anchorage-independent growth, invasion and migration. Blockade of AxI function abrogated phosphorylation of ERBB2 (Her-2/neu) at the Tyr877 residue, indicative of receptor crosstalk. AxI RTK is an adverse prognostic factor in esophageal adenocarcinoma. The availability of small molecule inhibitors of AxI function is useful for the prevention or treatment of esophageal adenocarcinoma.

Selection of a Treatment Method

After a subject is diagnosed as having Barrett esophagus or esophageal adenocarcinoma a method of treatment is selected. Because Barrett esophagus may progress to esophageal adenocarcinoma, therapies often involve treatment to slow, block, or otherwise prevent progression to esophageal adenocarcinoma. In other embodiments, a therapy for Barrett esophagus may involve monitoring the disease. Methods of treating subjects having levels of AxI consistent with a diagnosis of non-dysplastic Barrett epithelium or low-grade dysplasia are less aggressive than those used to treat subjects having levels of AxI consistent with esophageal adenocarcinoma. In such subjects, therapeutic regimens are selected that may be less aggressive and have fewer adverse side effects. Methods of the invention are also useful in selecting a treatment for a subject that has esophageal adenocarcinoma. Such subjects are treated with more aggressive treatment regimens than subjects with Barrett esophagus. In one embodiment, AxI is useful for identifying subjects in need of surgery for the treatment of esophageal

adenocarcinoma, and for identifying subjects at risk of having or having a propensity to develop metastatic disease. Such subjects could benefit from the most aggressive therapies where the benefit of therapy outweighs the risk of adverse side-effects.

Thus, the invention provides methods for selecting an appropriate therapy for a subject, the method involving identifying a subject as having esophageal adenocarcinoma, and administering to the subject a therapeutic treatment appropriate for that disease. Exemplary treatments include R428 alone or in combination with lapatinib.

Inhibitory Nucleic Acids

Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of a AxI polypeptide. Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes a AxI polypeptide (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to a AxI polypeptide to modulate its biological activity (e.g., aptamers).

Ribozymes

Catalytic RNA molecules or ribozymes that include an antisense AxI sequence of the present invention can be used to inhibit expression of a AxI nucleic acid molecule in vivo. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA- specific ribozymes is described in Haseloff et al., Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 Al , each of which is incorporated by reference.

Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8:183, 1992. Example of hairpin motifs are described by Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

Small hairpin RNAs consist of a stem-loop structure with optional 3' UU-overhangs.

While there may be variation, stems can range from 21 to 31 bp (desirably 25 to 29 bp), and the loops can range from 4 to 30 bp (desirably 4 to 23 bp). For expression of shRNAs within cells, plasmid vectors containing either the polymerase ITf Hl-RNA or U6 promoter, a cloning site for the stem-looped RNA insert, and a 4-5-thymidine transcription termination signal can be employed. The Polymerase III promoters generally have well-defined initiation and stop sites and their transcripts lack poly(A) tails. The termination signal for these promoters is defined by the polythymidine tract, and the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed shRNA, which is similar to the 3' overhangs of synthetic siRNAs. Additional methods for expressing the shRNA in mammalian cells are described in the references cited above.

siRNA

Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down- regulating gene expression (Zamore et al., Cell 101 : 25-33; Elbashir et al., Nature 41 1 : 494-498, 2001 , hereby incorporated by reference). The therapeutic effectiveness of an sirNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39.2002).

Given the sequence of a target gene, siRNAs may be designed to inactivate that gene.

Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of the AxI gene can be used to design small interfering RNAs (siRNAs). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat a vascular disease or disorder.

The inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of AxI expression. In one embodiment, AxI expression is reduced in an endothelial cell or an astrocyte. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001 ; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251 , 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of- function phenotypes in mammalian cells.

In one embodiment of the invention, double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550- 553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.

Small hairpin RNAs consist of a stem-loop structure with optional 3' UU-overhangs. While there may be variation, stems can range from 21 to 31 bp (desirably 25 to 29 bp), and the loops can range from 4 to 30 bp (desirably 4 to 23 bp). For expression of shRNAs within cells, plasmid vectors containing either the polymerase III Hl-RNA or U6 promoter, a cloning site for the stem-looped RNA insert, and a 4-5-th ymidine transcription termination signal can be employed. The Polymerase III promoters generally have well-defined initiation and stop sites and their transcripts lack poly(A) tails. The termination signal for these promoters is defined by the polythymidine tract, and the transcript is typically cleaved after the second undine. Cleavage at this position generates a 3' UU overhang in the expressed shRNA, which is similar to the 3' overhangs of synthetic siRNAs. Additional methods for expressing the shRNA in mammalian cells are described in the references cited above.

Delivery of Nucleobase Oligomers

Naked inhibitory nucleic aicd molecules, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,61 1, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).

In one embodiment, the invention provides methods of treating Barrett esophagus or esophageal adenocarcinoma featuring a polynucleotide encoding an inhibitory nucleic acid molecule that targets AxI is another therapeutic approach for treating a Barrett esophagus or esophageal adenocarcinoma. Expression of such inhibitory nucleic acid molecules in a neoplastic cell is expected to be useful for ameliorating the disease. Such nucleic acid molecules can be delivered to cells of a subject having Barrett esophagus or esophageal adenocarcinoma. The nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of a inhibitory nucleic acid molecule or fragment thereof can be produced.

Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997). For example, a polynucleotide encoding a AxI inhibitory nucleic acid molecule, variant, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1 :55-61, 1990; Sharp, The Lancet 337: 1277- 1278, 1991 ; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:31 1-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991 ; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). Most preferably, a viral vector is used to administer a CHOP or ATF-4 polynucleotide systemically.

Non-viral approaches can also be employed for the introduction of therapeutic to a cell of a patient requiring inhibition of a neoplasia or induction of cell death in a neoplasia. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989;

Staubinger et al., Methods in Enzymology 101 :512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247: 1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.

Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue.

The expression of an inhibitory nucleic acid molecule in a cell can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers.

Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

The dosage of the administered inhibitory nucleic acid molecule depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Anti-Axl Therapeutic Conjugates

The AxI polypeptide provides a cell surface target for cell-specific delivery of therapeutics (e.g., chemotherapeutic agents, toxins, and radioactive isotope). By specifically targeting AxI expressing cells, the invention reduces adverse side-effects, and provides for the delivery of therapeutics to cells (e.g., esophageal adenocarcinoma cells and Barrett esophagus cells) that display AxI on their surface. In one embodiment, an anti-Axl antibody, aptamer, or other AxI binding agent is conjugated to one or more of the following toxins or active fragments thereof: diphtheria toxin, Clostridium perfringens enterotoxin, exotoxin A chain, ricin, abrin A chain, modeccin A chain, alpha-sarcin, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

Chemotherapeutic agents useful in the generation of anti-Axl antibody, aptamer, or other AxI binding agent conjugates include: DNA damaging agents, inhibitors of microtubule polymerization or depolymerization and antimetabolites. In particular, the invention provides conjugates of AxI and any of the following: methotrexate, methopterin, dichloromethotrexate, 5- fluorouracil, 6-mercaptopurine, cytosine arabinoside, gemcitabine, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, morpholino-doxorubicin, l-(2-choroehthyl)-l,2-dimethanesulfonyl hydrazide, aminopterin methopterin, esperamicin, mitomycin C, mitomycin A, actinomycin, bleomycin, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere, retinoic acid, butyric acid, camptothecin, calicheamicin, bryostatins, cephalostatins, ansamitocin, actosin,

maytansinoids, dolostatins, and cephalostatins. An anti-Axl antibody, aptamer, or other AxI binding agent or fragment thereof, may be conjugated using any linking groups known in the art for making therapeutic agent conjugates. Such linking groups include disufide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups. Conjugates of the AxI antibody and a therapeutic agent may be made using, for example, a bifunctibnal protein coupling agent (e.g., N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For selective destruction of an Axl-expressing tumors, the anti-Axl antibody, aptamer, or other AxI binding agent may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-Axl antibodies, for example. Examples include At ²", I¹³¹, 1.¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example I¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine- 123, iodine-131, indium- 11 1, fluorine- 19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine- 19 in place of hydrogen. Labels can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine- 123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyO-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon- 14- labeled 1 -isothiocyanatobenzyl-S-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/1 1026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfϊde-containing linker (Chari et al. Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Pharmaceutical Therapeutics

For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically- acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a therapeutic identified herein in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the neoplasia. Generally, amounts will be in the range of those used for other agents used in the treatment of other diseases associated with neoplasia, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that is cytotoxic to a neoplastic cell, that reduces AxI expression or biological activity, or that reduces the proliferation, survival, or invasiveness of a neoplastic cell as determined by a method known to one skilled in the art, or using any that assay that measures the expression or the biological activity of a AxI polypeptide.

Formulation of Pharmaceutical Compositions

The administration of a compound for the treatment of a neoplasia may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing a neoplasia. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1 -95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g.,

subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R.

Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical

Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 1 μg compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight. In other embodiments this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 mg/Kg body weight. In other embodiments, it is envisaged that doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.

Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with the thymus; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a neoplasia by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type (e.g., neoplastic cell). For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner.

Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non- toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a neoplasia, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable active antineoplastic therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.

Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2- hydroxyethyl-L-glutaminine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be nonbiodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Solid Dosage Forms For Oral Use

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate,

carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose,

carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or

polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl

methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material, such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active anti-neoplasia therapeutic substance). The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical Technology, supra.

At least two anti-neoplasia therapeutics may be mixed together in the tablet, or may be partitioned. In one example, the first active anti-neoplasia therapeutic is contained on the inside of the tablet, and the second active anti-neoplasia therapeutic is on the outside, such that a substantial portion of the second anti-neoplasia therapeutic is released prior to the release of the first anti-neoplasia therapeutic.

Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Controlled Release Oral Dosage Forms

Controlled release compositions for oral use may, e.g., be constructed to release the active anti-neoplasia therapeutic by controlling the dissolution and/or the diffusion of the active substance. Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol

palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1 ,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated metylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.

A controlled release composition containing one or more therapeutic compounds may also be in the form of a buoyant tablet or capsule (i.e., a tablet or capsule that, upon oral administration, floats on top of the gastric content for a certain period of time). A buoyant tablet formulation of the compound(s) can be prepared by granulating a mixture of the compound(s) with excipients and 20-75% w/w of hydrocolloids, such as hydroxyethylcellulose,

hydroxypropylcellulose, or hydroxypropylmethylcellulose. The obtained granules can then be compressed into tablets. On contact with the gastric juice, the tablet forms a substantially water- impermeable gel barrier around its surface. This gel barrier takes part in maintaining a density of less than one, thereby allowing the tablet to remain buoyant in the gastric juice. Diagnostics

Levels of AxI have been correlated with the progression of Barrett esophagus to esophageal adenocarcinoma, and thus are useful in diagnosing this condition. In particular, low levels of AxI polynucleotides and/or polypeptide are useful for distinguishing non-dysplastic Barrett epithelium and low-grade dysplasia from high-grade dysplasia. In other embodiments, increased levels of AxI are indicative of esophageal adenocarcinoma. In one embodiment, levels are increased by at least about 3-fold, 5-fold, or even by as much as 10-fold or more. A variety of protocols for measuring an alteration in the expression AxI expression are known, including immunological methods (such as ELISAs and RIAs), mass spectrometric approaches, immunohistochemical analysis on tissues, flow cytometric analysis, real time quantitative RT- PCR for AxI transcripts, gene expression microarrays, next generation genomic and expression profiling (such as with the Illumina Genome Analyzer or Applied Biosystems SOLid), and fluorescent in situ hybridization (FISH) for AxI gene dosage. Such methods provide a basis for diagnosing esophageal adenocarcinoma. As described herein, increases in AxI levels in clinical samples indicate that the presence or absence of a esophageal adenocarcinoma, a particular stage of disease progression from Barrett esophagus to esophageal adenocarcinoma, indicate a good or poor prognosis, or indicate the efficacy of drug treatment.

The invention provides methods for aiding esophageal adenocarcinoma diagnosis using AxI levels, as specified herein. AxI is differentially present in samples of a subject having or having a propensity to develop esophageal adenocarcinoma and a healthy control subject in whom Barrett esophagus or esophageal adenocarcinoma is not present. For example, AxI is expressed at an elevated level in Barrett esophagus and in esophageal adenocarcinoma subjects than in normal subjects. Therefore, detection of AxI in a sample from a subject would provide useful information regarding the probability that the person may have Barrett esophagus or esophageal adenocarcinoma.

The detection of AxI is then correlated with a probable diagnosis of Barrett esophagus or esophageal adenocarcinoma. In some embodiments, the detection of the mere presence or absence of AxI polypeptide or polynucleotide, without quantifying the amount thereof, is useful and can be correlated with a probable diagnosis of Barrett esophagus or esophageal

adenocarcinoma. The measurement of AxI may also involve quantifying AxI to correlate the detection of AxI with a probable diagnosis of esophageal adenocarcinoma. Thus, if the amount of the AxI detected in a subject being tested is different compared to a control amount (i.e., higher than the healthy control and/or higher than the amount present in Barrett esophagus), then the subject being tested has a higher probability of having esophageal adenocarcinoma.

The correlation may take into account the amount of AxI in the sample compared to a control amount of AxI in normal subjects or in subjects that have Barrett esophagus. A control can be, e.g., the average or median amount of AxI present in comparable samples of normal subjects or in subjects having Barrett esophagus. The control amount is measured under the same or substantially similar experimental conditions as in measuring the test amount. As a result, the control can be employed as a reference standard, where the normal phenotype is known, and each result can be compared to that standard, rather than re-running a control.

Accordingly, the AxI level may be obtained from a subject sample and compared to a reference level obtained from a reference population, so that it is possible to classify the subject as having Barrett esophagus or esophageal adenocarcinoma. As is well understood in the art, the techniques used to detect AxI can be adjusted to increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician.

Optionally, methods described herein may be combined with any conventional method for the diagnosis of esophageal adenocarcinoma (e.g., endoscopic biopsy with accompanying histological examination, endoscopic ultrasound, computerized axial tomography).

Anti-Axl Diagnostic Conjugates

Labeled anti-Axl antibodies, aptamers, and other agents that specifically bind to AxI can be conjugated to detectable moieties. The anti-Axl conjugates can be used to detect, diagnose, or monitor Barrett esophagus or esophageal adenocarcinoma, as well as other neoplasia that express or express increased levels of AxI relative to healthy control tissues. The presence of AxI or a relatively high amount of AxI transcript or AxI polypeptide in tissue of a subject may indicate a predisposition for the development of esophageal adenocarcinoma, or may provide a means for detecting esophageal adenocarcinoma prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of Barrett esophagus.

Barrett esophagus and esophageal adenocarcinoma are associated with increased expression of AxI. In one embodiment, diagnosis comprises: (a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a mammal an effective amount of a labeled anti- AxI antibody or other agent that specifically binds to the AxI molecule, respectively; (b) waiting for a time interval following the administering to allow the labeled molecule to preferentially concentrate at sites in the subject where the AxI molecule is expressed (and for unbound labeled molecule to be cleared to background level); (c) determining background level; and (d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with expression of AxI. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

Various in vivo assays are available to the skilled practitioner. For example, one can expose cells within the body of the mammal to an anti-Axl antibody, aptamer, or other AxI binding agent that is labeled with a detectable label, e.g., a radioactive isotope. Binding of the anti-Axl antibody, aptamer, or other AxI binding agent to cells in the mammal is evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a mammal previously exposed to the antibody.

Anti-Axl Therapeutic Conjugates

The AxI polypeptide provides a cell surface target for cell-specific delivery of therapeutics (e.g., chemotherapeutic agents, toxins, and radioactive isotope). By specifically targeting AxI expressing cells, the invention reduces adverse side-effects, and provides for the delivery of therapeutics to cells (e.g., esophageal adenocarcinoma cells and Barrett esophagus cells) that display AxI on their surface.

In one embodiment, an anti-Axl antibody, aptamer, or other AxI binding agent is conjugated to one or more of the following toxins or active fragments thereof: diphtheria toxin, Clostridium perfringens enterotoxin, exotoxin A chain, ricin, abrin A chain, modeccin A chain, alpha-sarcin, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

maytansinoids, dolostatins, and cephalostatins.

An anti-Axl antibody, aptamer, or other AxI binding agent or fragment thereof, may be conjugated using any linking groups known in the art for making therapeutic agent conjugates. Such linking groups include disufϊde groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups. Conjugates of the AxI antibody and a therapeutic agent may be made using, for example, a bifunctional protein coupling agent (e.g., N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N- maleimidomethyl)cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5- difluoro-2,4-dinitrobenzene).

For selective destruction of an Axl-expressing tumors, the anti-Axl antibody, aptamer, or other AxI binding agent may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-Axl antibodies, for example. Examples include At ²", I¹³¹, 1.¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example I¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine- 123, iodine-131, indium- 1 1 1 , fluorine- 19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.

Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis- active fluorine compounds (such as l ,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-14- labeled 1 -isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/1 1026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52: 127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Monitoring

Methods of characterizing Barrett esophagus or esophageal adenocarcinoma in a subject are also useful in managing subject treatment based on the subject's status. The invention provides for such methods where AxI is measured before, during and/or after subject

management. In these cases, the methods are used to monitor the status of non-dysplastic Barrett epithelium and/or low-grade dysplasia, to monitor the progression of Barrett esophagus to esophageal adenocarcinoma, and/or to monitor the response of Barrett esophagus or esophageal adenocarcinoma to treatment. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a patient. Therapeutics that reduce levels of AxI to correspond to levels present in a healthy control subject are taken as particularly useful in the invention.

Detection Methods

The invention provides methods of detecting AxI in an esophageal tissue sample, lymph node sample, or other biological sample including any suspected organ site of spread or metastases, and in biological fluid samples such as blood, plasma, serum, urine, saliva, and esophageal lavage obtained from a subject. AxI polypeptide or polynucleotide expression is measured by procedures well known in the art, such as immunoassays (e.g., enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)), Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or Axl-specific antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), microarray analysis, or colorimetric assays, such as the Bradford Assay and Lowry Assay. According to one specific embodiment, AxI polypeptide expression is determined in a diagnostic or prognostic assay by evaluating levels of AxI present on the surface of a cell using anti- AxI antibodies. Alternatively, or additionally, one can measure levels of AxI polypeptide-encoding nucleic acid or mRNA in the cell, e.g., via fluorescent in situ hybridization using a nucleic acid based probe corresponding to a AxI- encoding nucleic acid or the complement thereof; (FISH; see WO98/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR).

The sequence of AxI polypeptides and polynucleotides are known in the art and can be identified in public databases by searching on the gene or polypeptide name. For example, the human AxI polypeptide is NCBI Reference Sequence: P30530. The amino acid sequence of an exemplary AxI polypeptide is provided below.

1 mawrcprmgr vplawclalc gwacmaprgt qaeespfvgn pgnitgargl tgtlrcqlqv

61 qgeppevhwl rdgqilelad stqtqvplge deqddwivvs qlritslqls dtgqyqclvf 121 lghqtfvsqp gyvgleglpy f leepedrtv aantpfnlsc qaqgppepvd llwlqdavpl

181 atapghgpqr slhvpglnkt ssfsceahna kgvttsrtat itvlpqqprn lhlvsrqpte

241 levawtpgls giyplthctl qavlsndgmg iqagepdppe epltsqasvp phqlrlgslh

301 phtpyhirva ctssqgpssw thwlpvetpe gvplgppeni satrngsqaf vhwqeprapl

361 qgtllgyrla yqgqdtpevl mdiglrqevt lelqgdgsvs nltvcvaayt aagdgpwslp 421 vpleawrpgq aqpvhqlvke pstpafswpw wyvllgavva aacvlilalf lvhrrkketr

481 ygevfeptve rgelvvryrv rksysrrtte atlnslgise elkeklrdvm vdrhkvalgk

541 tlgegefgav megqlnqdds ilkvavktmk iaictrsele dflseavcmk efdhpnvmrl

601 igvcfqgser esfpapvvil pfmkhgdlhs fllysrlgdq pvylptqmlv kfmadiasgm

661 eylstkrfih rdlaarncml nenmsvcvad fglskkiyng dyyrqgriak mpvkwiaies 721 ladrvytsks dvwsfgvtmw eiatrgqtpy pgvenseiyd ylrqgnrlkq padcldglya

781 lmsrcwelnp qdrps ftelr edlentlkal ppaqepdeil yvnmdegggy peppgaagga

841 dpptqpdpkd scscltaaev hpagryvlcp sttpspaqpa drgspaapgq edga

An AxI polynucleotide is a nucleic acid molecule that encodes an AxI polypeptide. An exemplary AxI nucleic acid molecule is NCBI Accession No.: NM_001699, which sequence is provided below:

1 gggaaggagg caggggtgct gagaaggcgg ctgctgggca gagccggtgg caagggcctc

61 ccctgccgct gtgccaggca ggcagtgcca aatccgggga gcctggagct ggggggaggg

121 ccggggacag cccggccctg ccccctcccc cgctgggagc ccaacaactt ctgaggaaag 181 tttggcaccc atggcgtggc ggtgccccag gatgggcagg gtcccgctgg cctggtgctt

241 ggcgctgtgc ggctgggcgt gcatggcccc caggggcacg caggctgaag aaagtccctt

301 cgtgggcaac ccagggaata tcacaggtgc ccggggactc acgggcaccc ttcggtgtca

361 gctccaggtt cagggagagc cccccgaggt acattggctt cgggatggac agatcctgga

421 gctcgcggac agcacccaga cccaggtgcc cctgggtgag gatgaacagg atgactggat 481 agtggtcagc cagctcagaa tcacctccct gcagctttcc gacacgggac agtaccagtg

541 tttggtgttt ctgggacatc agaccttcgt gtcccagcct ggctatgttg ggctggaggg

601 cttgccttac ttcctggagg agcccgaaga caggactgtg gccgccaaca cccccttcaa

661 cctgagctgc caagctcagg gacccccaga gcccgtggac ctactctggc tccaggatgc

721 tgtccccctg gccacggctc caggtcacgg cccccagcgc agcctgcatg ttccagggct 781 gaacaagaca tcctctttct cctgcgaagc ccataacgcc aagggggtca ccacatcccg

841 cacagccacc atcacagtgc tcccccagca gccccgtaac ctccacctgg tctcccgcca

901 acccacggag ctggaggtgg cttggactcc aggcctgagc ggcatctacc ccctgaccca

961 ctgcaccctg caggctgtgc tgtcagacga tgggatgggc atccaggcgg gagaaccaga

1021 ccccccagag gagcccctca cctcgcaagc atccgtgccc ccccatcagc ttcggctagg 1081 cagcctccat cctcacaccc cttatcacat ccgcgtggca tgcaccagca gccagggccc

1141 ctcatcctgg acccactggc ttcctgtgga gacgccggag ggagtgcccc tgggcccccc

1201 tgagaacatt agtgctacgc ggaatgggag ccaggccttc gtgcattggc aagagccccg

1261 ggcgcccctg cagggtaccc tgttagggta ccggctggcg tatcaaggcc aggacacccc

1321 agaggtgcta atggacatag ggctaaggca agaggtgacc ctggagctgc agggggacgg 1381 gtctgtgtcc aatctgacag tgtgtgtggc agcctacact gctgctgggg atggaccctg

1441 gagcctccca gtacccctgg aggcctggcg cccagtgaag gaaccttcaa ctcctgcctt

1501 ctcgtggccc tggtggtatg tactgctagg agcagtcgtg gccgctgcct gtgtcctcat

1561 cttggctctc ttccttgtcc accggcgaaa gaaggagacc cgttatggag aagtgtttga

1621 accaacagtg gaaagaggtg aactggtagt caggtaccgc gtgcgcaagt cctacagtcg 1681 tcggaccact gaagctacct tgaacagcct gggcatcagt gaagagctga aggagaagct

1741 gcgggatgtg atggtggacc ggcacaaggt ggccctgggg aagactctgg gagagggaga

1801 gtttggagct gtgatggaag gccagctcaa ccaggacgac tccatcctca aggtggctgt 1861 gaagacgatg aagattgcca tctgcacgag gtcagagctg gaggatttcc tgagtgaagc 1921 ggtctgcatg aaggaatttg accatcccaa cgtcatgagg ctcatcggtg tctgtttcca 1981 gggttctgaa cgagagagct tcccagcacc tgtggtcatc ttacctttca tgaaacatgg 2041 agacctacac agcttcctcc tctattcccg gctcggggac cagccagtgt acctgcccac 2101 tcagatgcta gtgaagttca tggcagacat cgccagtggc atggagtatc tgagtaccaa 2161 gagattcata caccgggacc tggcggccag gaactgcatg ctgaatgaga acatgtccgt 2221 gtgtgtggcg gacttcgggc tctccaagaa gatctacaat ggggactact accgccaggg 2281 acgtatcgcc aagatgccag tcaagtggat tgccattgag agtctagctg accgtgtcta 23.41 caccagcaag agcgatgtgt ggtccttcgg ggtgacaatg tgggagattg ccacaagagg 2401 ccaaacccca tatccgggcg tggagaacag cgagatttat gactatctgc gccagggaaa 2461 tcgcctgaag cagcctgcgg actgtctgga tggactgtat gccttgatgt cgcggtgctg 2521 ggagctaaat ccccaggacc ggccaagttt tacagagctg cgggaagatt tggagaacac 2581 actgaaggcc ttgcctcctg cccaggagcc tgacgaaatc ctctatgtca acatggatga 2641 gggtggaggt tatcctgaac cccctggagc tgcaggagga gctgaccccc caacccagcc 2701 agaccctaag gattcctgta gctgcctcac tgcggctgag gtccatcctg ctggacgcta 2761 tgtcctctgc ccttccacaa cccctagccc cgctcagcct gctgataggg gctccccagc 2821 agccccaggg caggaggatg gtgcctgaga caaccctcca cctggtactc cctctcagga 2881 tccaagctaa gcactgccac tggggaaaac tccaccttcc cactttccca ccccacgcct 2941 tatccccact tgcagccctg tcttcctacc tatcccacct ccatcccaga caggtccctc 3001 cccttctctg tgcagtagca tcaccttgaa agcagtagca tcaccatctg taaaaggaag 3061 gggttggatt gcaatatctg aagccctccc aggtgttaac attccaagac tctagagtcc 3121 aaggtttaaa gagtctagat tcaaaggttc taggtttcaa agatgctgtg agtctttggt 3181 tctaaggacc tgaaattcca aagtctctaa ttctattaaa gtgctaaggt tctaaggcct 3241 actttttttt tttttttttt tttttttttt tttgcgatag agtctcactg tgtcacccag 3301 gctggagtgc agtggtgcaa tctcgcctca ctgcaacctt cacctaccga gttcaagtga 3361 ttttcctgcc ttggcctccc aagtagctgg gattacaggt gtgtgccacc acacccggct 3421 aatttttata tttttagtag agacagggtt tcaccatgtt ggccaggctg gtctaaaact 3481 cctgacctca agtgatctgc ccacctcagc ctcccaaagt gctgagatta caggcatgag 3541 ccactgcact caaccttaag acctactgtt ctaaagctct gacattatgt ggttttagat 3601 tttctggttc taacattttt gataaagcct caaggtttta ggttctaaag ttctaagatt 3661 ctgattttag gagctaaggc tctatgagtc tagatgttta ttcttctaga gttcagagtc 3721 cttaaaatgt aagattatag attctaaaga ttctatagtt ctagacatgg aggttctaag 3781 gcctaggatt ctaaaatgtg atgttctaag gctctgagag tctagattct ctggctgtaa 3841 ggctctagat cataaggctt caaaatgtta tcttctcaag ttctaagatt ctaatgatga 3901 tcaattatag tttctgaggc tttatgataa tagattctct tgtataagat cctagatcct 3961 aagggtcgaa agctctagaa tctgcaattc aaaagttcca agagtctaaa gatggagttt 4021 ctaaggtccg gtgttctaag atgtgatatt ctaagactta ctctaagatc ttagattctc 4081 tgtgtctaag attctagatc agatgctcca agattctaga tgattaaata agattctaac 4141 ggtctgttct gtttcaaggc actctagatt ccattggtcc aagattccgg atcctaagca 4201 tctaagttat aagactctca cactcagttg tgactaacta gacaccaaag ttctaataat 4261 ttctaatgtt ggacaccttt aggttctttg ctgcattctg cctctctagg accatggtta 4321 agagtccaag aatccacatt tctaaaatct tatagttcta ggcactgtag ttctaagact 4381 caaatgttct aagtttctaa gattctaaag gtccacaggt ctagactatt aggtgcaatt 4441 tcaaggttct aaccctatac tgtagtattc tttggggtgc ccctctcctt cttagctatc 4501 attgcttcct cctccccaac tgtgggggtg tgcccccttc aagcctgtgc aatgcattag 4561 ggatgcctcc tttcccgcag gggatggacg atctcccacc tttcgggcca tgttgccccc 4621 gtgagccaat ccctcacctt ctgagtacag agtgtggact ctggtgcctc cagaggggct 4681 caggtcacat aaaactttgt atatcaacga aaaaaa In another embodiment, the sequence of a longer splice variant, NM_021913 is used.

One of skill in the art will recognize that any suitable method can be used to detect the AxI polypeptides described herein. Successful practice of the invention can be achieved with one or a combination of methods that can detect and/or quantify AxI. Such methods include, without limitation, hybridization-based methods including those employed in microarrays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry, atomic force microscopy, and 2- dimensional gel electrophoresis. Methods may further include, one or more of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS),

desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI- MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), quadrupole mass spectrometry, fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry.

Diagnostic Kits

In one aspect, the invention provides kits for monitoring and diagnosing Barrett esophagus or esophageal adenocarcinoma, wherein the kits can be used to detect AxI. For example, the kits can be used to detect AxI differentially present in samples of Barrett esophagus or esophageal adenocarcinoma subjects vs. normal subjects. If desired, the kit further comprises reagents suitable for measuring AxI levels. The kits of the invention have many applications. For example, the kits can be used to distinguish between Barrett esophagus or esophageal adenocarcinoma and control, to determine if a subject has Barrett esophagus or esophageal adenocarcinoma or to determine that the subject does not have Barrett esophagus or esophageal adenocarcinoma, thus aiding in the diagnosis of these diseases. In other embodiments, the kits distinguish between non-dysplastic Barrett epithelium and/or low-grade dysplasia and esophageal adenocarcinoma.

The kits of the invention may include instructions for the assay, reagents, testing equipment (test tubes, reaction vessels, needles, syringes, etc.), standards for calibrating the assay, and/or equipment provided or used to conduct the assay. The instructions provided in a kit according to the invention may be directed to suitable operational parameters in the form of a label or a separate insert.

The kits may also include an adsorbent, wherein the adsorbent retains AxI, and written instructions for use of the kit for detection of Barrett esophagus or esophageal adenocarcinoma. Such a kit could, for example, comprise: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent is suitable for binding AxI, and (b) instructions to detect AxI by contacting a sample with the adsorbent and detecting the AxI polypeptide or polynucleotide retained by the adsorbent. Accordingly, the kit could further comprise a detection reagent.

Optionally, the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if the test amount of a marker detected in a sample is a diagnostic amount consistent with a diagnosis Barrett esophagus or esophageal adenocarcinoma.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Global transcriptomic profiling of epithelial cancers and their precursor lesions provides an unbiased opportunity for the identification of diagnostic and prognostic biomarkers, and can elucidate novel therapeutic targets. As described in more detail below, a "Long" Serial Analysis of Gene Expression (L-SAGE) libraries was carried out using a panel of mucosal biopsies obtained from a Barrett esophagus patient who developed esophageal adenocarcinoma during the course of endoscopic surveillance (Saha et al. Nat Biotechnol 2002; 20:508-12). "Tags" corresponding to the AxI receptor tyrosine kinase (RTK) were identified as being significantly and progressively upregulated during multistep esophageal carcinogenesis. AxI expression was identified as an independent adverse prognostic factor in surgically resected EAC. This RTK regulates multiple components of the neoplastic phenotype (such as invasion, migration, and in vivo engraftment) in EAC cell lines. The major intracellular effectors of AxI were characterized, including the identification of RaI GTPase proteins as novel effectors of the AxI signaling cascade, particularly in the context of regulating cell motility. The recent availability of small molecule pharmacological inhibitors of AxI function provides a unique opportunity for developing targeted therapies for advanced EAC.

Example 1: Progressive Upregulation of AxI and its Cognate Ligand Gas6 during Multistep Progression of Barrett Esophagus to EAC

In order to identify transcriptomic abnormalities during Barrett esophagus progression, four independent "long" SAGE (L-SAGE) libraries (Saha et al. Nat Biotechnol 2002; 20:508-12) were generated using RNA obtained from histologically validated mucosal biopsies of normal squamous epithelium (NSE), low-grade dysplasia (LGD), high-grade-dysplasia (HGD), and esophageal adenocarcinoma, all from a single individual on endoscopic surveillance (Figure IA and Table 1).

A total of 14,208 clones were sequenced in collaboration with the Cancer Genome Anatomy Project (CGAP). 302,942 L-SAGE tags were extracted from the raw sequence data, and those tags present more than once clustered into 21,542 independent transcripts (Table 2).

The tag information contained within the four L-SAGE libraries can be downloaded from the SAGE Genie website (http://cgap.nci.nih.gov/SAGE), using the Library Finder function.

Pair-wise comparisons were performed between the L-SAGE libraries to identify genes that had statistically significant differences in transcript levels during Barrett progression. As shown in Table 3, -900 to 2,400 tags were identified that were significantly altered in any given pair-wise comparison.

Not surprisingly, the greatest amplitude of difference was observed in comparisons with NSE, underscoring the biologically distinct nature of Barrett metaplastic epithelium. A subset of 932 L-SAGE tags were selected that were expressed at least once in the transcriptome of all four libraries, and cluster analysis with these tags unequivocally delineated the distinct histological categories of mucosal tissue used in our analysis (Figure IB, which represents a further sub- selection of the most differentially expressed tags). In Table 4, 22 unique L-SAGE tags (and corresponding annotated genes) are listed that meet two prerequisite characteristics: first, they exhibit maximal upregulation based on fold-change in pair-wise comparison between EAC and NSE libraries, at a P<0.05, and second, these tags demonstrate progressive upregulation in normalized counts from squamous epithelium through Barrett dysplasia to cancer.

Included in this differentially overexpressed list were transcripts encoding receptor tyrosine kinases (AXL), oncogene homologs (MAFF), nuclear receptors (VDR), heterochromatin associated non-histone proteins (CBX5), and major histocompatibility proteins (HLA-A, HLA-E, and CD74), amongst others. This stringently enriched list of upregulated transcripts provides biomarker useful in diagnosing esophageal carcinoma or a predisposition to develop esophageal carcinoma. It further provides a list of therapeutic targets associated with esophageal neoplasia. From the list of upregulated L-SAGE transcripts in Table 4, the AxI RTK was selected for further validation in tissue samples. This was based on the emerging association between AxI and human cancer (Gjerdrum et al., Proc Natl Acad Sci U S A 2010; 107: 1124-9; Li et al.

Oncogene 2009; 28:3442-55; Koorstra et al., Cancer Biol Ther 2009; 8:618-26; Linger et al., Adv Cancer Res 2008; 100:35-83) and the assumption that RTKs, in general, have emerged as the most promising therapeutic candidates in recent years (Zwick et al., Trends MoI Med 2002; 8: 17-23). Immunohistochemical analysis for AxI protein expression was performed on an independent set of archival tissue microarray samples, representing the entire histological spectrum from metaplastic Barrett esophagus to adenocarcinoma, and comprised of 292 independent tissue "cores" from 92 patients with surgically resected EAC (Figure 2A). Minimal AxI expression was observed in esophageal squamous epithelium, gastric cardiac mucosa, non- dysplastic Barrett mucosa and in LGD, with significant upregulation of expression observed in samples of HGD, EAC, and associated lymph node metastases (P<0.05, Figure 2B). When the EACs per se were stratified by level of AxI staining, 56 of 92 (61%) cases demonstrated high levels of expression. Amongst a list of clinicopathological variables, AxI overexpression was significantly associated with depth of invasion (P = 0.007) and with T stage in the TNM classification (P = 0.027) (Table 5).

In this cohort of 92 EACs, AxI overexpression was significantly associated with reduced median survival (P = 0.004) on univariate analysis, along with T stage and presence of metastases

(Figure 1C and Table 6).

Of note, AxI expression was also an independent predictor of median survival on multivariate analysis (P = 0.036, Table 7).

These findings in a relatively large cohort of EACs, and associated precursor lesions, underscore a putative role for AxI in the progression from Barrett mucosa to adenocarcinoma, as well as in imparting an adverse prognosis to the subset of cases with aberrant expression. In order to confirm whether AxI receptor upregulation is accompanied by concomitant overexpression of the activating ligand, immunohistochemistry for Gas6 was performed in serial sections of the identical TMAs used above. The pattern of Gas6 expression was essentially comparable to that of AxI, with maximal upregulation occurring at the stage of invasive cancer and lymph node metastases (Figure 3A demonstrates Gas6 labeling in a single patient along the multistep progression of Barrett esophagus to EAC; panel B is the summary for all 292 independent cores). In contrast to AxI however, no impact of Gas6 expression levels in EACs was found upon median survival (Figure 3C). Gas6 upregulation has previously been implicated as a negative prognostic influence in solid cancers. To determine whether expression of its cognate receptor in tissues might be a limiting factor, 5he 92 EACs were categorized into four sub-cohorts, based on the independent quantification of Gas6 ligand and AxI receptor expression, respectively (Figure 3D). Kaplan-Meier survival estimates demonstrated that, even in cancers with high Gas6 ligand expression, AxI remained a limiting factor in influencing survival, with a persistent significant difference in outcome between cases with "high" and "low" receptor levels.

Example 2: AxI regulates multiple facets of the transformed phenotype in EAC cell lines

The level of expression of AxI and its cognate ligand, Gas6,¹⁰' " was assessed in a panel of non-neoplastic esophageal keratinocytes (NEK2 and HEEPIC) and EAC cell lines (OE33 and JH-EsoAdl), as a prelude to functional studies. AxI protein was overexpressed in both EAC lines compared to the esophageal keratinocytes (Figure 4A). In light of the striking AxI overexpression in JH-EsoAdl cells, genome-wide copy number analysis was performed using 244K microarrays (Agilent), which revealed a chromosome 19ql 3.1 amplification encompassing the AXL gene locus (Figure 5); copy number gain in JH-EsoAdl cells was confirmed by genomic qPCR and by fluorescence in situ hybridization. Pronounced Gas6 expression was also seen in the JH-EsoADl cells, suggesting the existence of an autocrine and / or paracrine ligand- receptor feedback loop in these cells. The question of whether knockdown of AxI function in EAC lines would impact upon their transformed phenotype was examined. JH-EsoADl and OE33 cells were stably infected with lentiviral vectors expressing AXL short hairpin RNA (shRNA) Table 8 vide infra, TRCN0000000575), and near-complete knockdown of transcript and protein was confirmed in both cell lines (Figure 4B-C). No inhibition of in vitro cell viability was observed in either cell line upon AxI blockade; in contrast, modified Boyden chamber assays demonstrated significant impairment of invasion and migration in both JH- EsoAdl and OE33 clones expressing AXL shRNA, compared to the respective scrambled shRNA controls (Figures 6A-B and 2C-D, respectively). The deleterious impact of AxI knockdown on the transformed phenotype of EAC was further underscored by the significant inhibition of anchorage independent growth in OE33 cells (Figure 6E), and the complete failure of tumor engraftment in immunocompromised mice using JH-EsoAdl cells (Figure 6F).

Example 3: AxI regulates EAC cell motility through Ral-dependent mechanisms

Previously, multiple reports in cancer cells, as well as in non-neoplastic settings, have implicated the phosphatidylinositol-3-kinase (PI-3-kinase)/Akt and p42/p44 mitogen activated protein kinase (MAPK) pathways as effectors of AxI function. The status of activation of these two pathways was examined in JH-EsoAdl and OE33 cells versus the respective clones stably expressing an AXL shRNA. In each case, the pathways were assessed in either the presence or absence of the cognate AxI ligand, Gas6. Not unexpectedly, Gasό stimulation of the AxI receptor was accompanied by increased phosphorylation of Akt and Erkl/2 (as a measure of MAPK pathway activation) in both cell lines. The effects of AXL shRNA expression were, however, divergent between the two models. Thus, in the JH-EsoAdl cells, AxI knockdown completely eliminated Gas6-induced phosphorylation of Akt at the Ser⁴⁷³ residue, and modestly decreased Erkl/2 phosphorylation (Figure 7A). In contrast, the Gas6-induced activation of either pathway was, for the most part, unaffected in OE33 clones expressing AXL shRNA, likely due to the existence of other concurrent RTK-dependent upstream signals (Figure 7B).

Nonetheless, in light of the significant phenotypic effects of AxI knockdown on OE33, comparable to that observed in JH-EsoAdl cells {see above), the existence of an effector mechanism regulated by AxI that is independent of either PI-3-kinase/Akt or MAPK pathways was postulated.

In recent years, the RaI GTPase proteins have emerged as a key mediator of oncogenic effects downstream of growth factor- stimulated receptor activation. In particular contexts (for example, in the setting on oncogenic Ras), the two RaI GTPase isoforms - RaIA and RaIB - have been demonstrated to be the principal determinants of both cellular transformation and motility. Therefore, the effects of AxI knockdown on the activity of the RaI GTPase proteins was investigated. Exogenous Gas6 ligand failed to enhance the "baseline" levels of GTP-bound (i.e., active) RaI proteins, while addition of epidermal growth factor (EGF) markedly increased these levels in both JH-EsoAdl and OE33 cell lines. This result was not unexpected, as RaI proteins are well established EGF effectors. AxI knockdown strikingly inhibited this EGF- dependent activation of RaIA and RaIB (Figure 7C). One of the effector targets of RaI proteins is RaIBPl, which demonstrates GTPase activating protein (GAP) activity for the Rho family GTPase, Cdc42. In both JH-EsoAdl and OE33 cells with AxI knockdown, reduction in RaI activity in the presence of EGF was also accompanied by a profound decrease in GTP-bound Cdc42 protein (Figure 7D). In order to further corroborate that downregulation of RaI signaling is indeed the underlying cause of the observed phenotypic differences between EAC cells with and without endogenous AxI function, OE33 AXL shRNA clones were generated with stable co- expression of a constitutively active form (RIf-CAAX) of the RaI guanine exchange factor (RaI GEF), Rgl2 (Wolthuis et al., EMBO J 1997; 16:6748-61). RIf-CAAX expression restored RaIA- GTP levels in OE33 AXL shRNA clones to that observed in scrambled infected OE33 clones (Figure 7E). This restitution in RaI function was accompanied by partial, but statistically significant (/^><0.05), "rescue" of both migration and invasion phenotypes when compared to OE33 clones with AxI knockdown (Figure 7F). This is the first demonstration of a requirement for sustained AxI function in maintaining EGF-dependent RaI GTPase activation in human cancer, and provides a mechanistic link between AxI and the regulation of cell motility.

Example 4: A novel small molecule antagonist of AxI functions impedes invasion and anchorage independent growth of OE33 cells, and sensitizes them to lapatinib

R428 is a recently described selective small molecule inhibitor of AxI tyrosine kinase activity that can block systemic metastases and improve survival of orthotopic xenograft models of breast cancer (Holland et al., Cancer Res 2010; 70: 1544-54, which is incorporated herein by reference). In OE33 cells, R428 demonstrated dose-dependent and significant reduction in both anchorage independent growth in soft agar and in vitro invasion (Figure 8A), comparable to results obtained with genetic (shRNA-mediated knockdown) (see Figure 6). Multiple reports have suggested that upregulation of AxI expression might represent a resistance mechanism to targeted therapies in solid tumors (Mahadevan et al. Oncogene 2007; 26:3909-19; Hong et al. Cancer Lett 2008; 268:314-24; Liu et al., Cancer Res 2009; 69:6871-8). Particularly compelling is the recent data demonstrating that AxI activation is observed in breast cancer cells with resistance to ERBB2/Her-2ne« targeted therapies like lapatinib, and that concurrent inhibition of AxI activity renders these cells sensitive to lapatinib. The Tyr⁸⁷⁷ residue of ERBB2 correlates with, and provides a convenient readout of, the biological activity of the receptor (Bose et al., Exp Cell Res 2009; 315:649-58) A dose-dependent reduction in ERBB2 Tyr⁸⁷⁷ phosphorylation was found upon exposure to R428 (Figure 8B), which was mirrored by genetic abrogation of AxI function using shRNA (Figure 8C). Without wishing to be bound by theory, these results suggested the intriguing possibility of crosstalk between AxI and ERBB2 receptors. As an extrapolation of these results, co-treatment of OE33 cells with R428 sensitized them to lapatinib in vitro (the IC₅0 of single agent lapatinib was 5.5μM versus 0.4μM upon combination) (Figure 8D), and that comparable sensitization was observed using a genetic strategy for AxI knockdown as well (Figure 8E).

The primary objective of this study was to perform transcriptomic profiling of Barrett esophagus-associated EAC, in order to identify candidate biomarkers and therapeutic targets for this malignancy. There are several unique facets to our study design that deserve comment. This is the first study to create SAGE profiles from non-invasive dysplastic lesions (LGD and HGD) that precede invasive EAC. The availability of these preneoplastic libraries

(http://cgap.nci.nih.gov/SAGE) should greatly facilitate future biomarker discovery efforts in Barrett esophagus. Second, the global profiling experiments described herein were performed on endoscopic mucosal biopsies obtained from a single patient who progressed to cancer during surveillance, rather than in mismatched tissues from disparate individuals. Using material from one patient provides a unique perspective on multistep Barrett esophagus progression, devoid of confounding influences due to inter-individual variations. This is also the first SAGE analysis in this disease model that utilizes a 17-bp Long-SAGE (L-SAGE) strategy, rather than the

"conventional" 10-bp approach (Short-S AGE). L-SAGE provides a higher specificity and fewer false positive results in tag-to-gene mapping compared to Short-SAGE. Finally, this study validates the use of minute endoscopic biopsies (~2-3mm in greatest diameter) as a feasible substrate for library construction and large scale nucleic acid profiling. With the ever decreasing costs of next generation sequencing, it is likely that this approach will be extended to larger numbers of biopsy samples, in the context of genomic, epigenetic or transcriptomic profiling. Bioinformatics analyses of the SAGE libraries identified the L-SAGE tag corresponding to the AXL gene transcript as being progressively upregulated during the transition from NSE through dysplasia to EAC. AxI is a member of the Tyro-3, AxI, and Mer (TAM) family of RTKs, which has been implicated in a diverse array of physiological functions such as cell adhesion, cell motility, proliferation, and regulation of inflammation (Linger et al., Adv Cancer Res 2008; 100:35-83). AxI was originally isolated as a transforming gene from human leukemia cells (O'Bryan et al., MoI Cell Biol 1991 ; 11 :5016-31). Since then, aberrant expression of AxI has been reported in a number of solid tumors, including pancreatic and breast cancers, and gliomas, amongst others (Gjerdrum et al., Proc Natl Acad Sci U S A 2010; Koorstra et al., Cancer Biol Ther 2009; 8:618-26. Linger et al., Adv Cancer Res 2008; 100:35-83; Holland et al., Cancer Res 2010; 70: 1544-54; Zhang et al. Cancer Res 2008; 68: 1905-15; Vajkoczy et al. Proc Natl Acad Sci U S A 2006; 103:5799-804. Based on this known association with human cancer, and the general propensity for RTKs to emerge as tangible therapeutic targets in oncology, AxI was selected for further validation in EAC. In a relatively large cohort of 92 surgically resected EAC patients, AxI expression in the neoplastic cells was significantly associated with depth of invasion, T stage, and decreased median survival. AxI also retained its independent adverse prognostic impact on survival on multivariate analysis, underscoring the biological relevance of cancer-specific overexpression. In the multistep progression of Barrett esophagus, significant AxI upregulation was observed essentially at the stage of HGD and beyond, with minimal to no expression in either non-dysplastic Barrett epithelium or in LGD. This indicates that surface AxI expression on HGD or EAC should be targeted as a diagnostic biomarker (for example, using a fluorescent anti-Axl antibody or peptide), or for the localized delivery of ablative therapies without incidental adverse effects in the surrounding epithelium. In EAC cell lines, genetic knockdown of endogenous AxI mitigated multiple aspects of the transformed phenotype, such as cell motility (migration and invasion), anchorage independent growth, and engraftment in immunocompromised mice, reiterating the importance of sustained AxI function for tumor maintenance

In other studies, the PI-3-kinase / Akt and p42/p44 MAPK pathways have been implicated as principal intracellular effectors of AxI signaling in cancer cells, as well as in non- cancer settings. As reported herein, while Gas6-dependent enhanced phosphorylation of Akt Ser⁴⁷³ and p42/p44 was observed in both Axl-expressing EAC cell lines, knockdown of AxI failed to abrogate the activation of these effector pathways in OE33 cells, despite the obvious effects on phenotype. This suggested the existence of additional effector(s) of AxI signaling in cancer cells. Although much of the scientific literature on effectors of RTK signal transduction has focused on PI-3-kinase / Akt and MAPK, the RaI GTPase proteins (RaIA and RaIB) have emerged as important mediators of growth factor signaling in recent years. For example, epidermal growth factor (EGF) ligand stimulates lamellipodia formation and migration through Ras-dependent recruitment of RaIGEFs to the plasma membrane, with resulting activation of RaI GTPase proteins. Counter and colleagues have shown that genetic knockdown of the two RaI GTPase isoforms (RaIA and RaIB) in KRAS2-mutant pancreatic cancer cells can abrogate both tumor engraftment and experimental (tail vein) metastases in vivo (Lim et al., Cancer Cell 2005; 7:533-45; Lim et al., Curr Biol 2006; 16:2385-94;

One of the principal functions of RaI proteins is regulation of the multi-protein complex known as the exocyst, which, in turn, regulates diverse biological functions, such as maintenance of epithelial cell polarity, cell motility and cytokinesis. As is the case for other GTP-binding proteins in the Ras superfamily, the level of RaI activation corresponds to the level of GTP occupancy of the RaI proteins, which is proximally controlled by the action of guanine nucleotide exchange factors (RaIGEFs, which load GTP) and GTPase activating proteins (RaIGAPs, which promote GTP hydrolysis to GDP). Genetic knockdown of AxI leads to profound diminishing of GTP-bound (active) RaI proteins in both OE33 and JH-EsoAdl cells. Further, loss of RaI activity is paralleled by reduction in GTP-bound Cdc42, a Rho GTPase that is one of the best characterized effectors of RaI proteins, especially in the context of cell motility. Finally, a compelling evidence supporting a role for the RaI pathway as a mediator of AxI function comes from the "rescue" experiments performed using a constitutively active form (RIf- CAAX) of the RaI GEF, Rgl2. Our studies present the first evidence for RaI GTPase as an effector of AxI signal transduction, and provide a facile kinase target for therapeutic inhibition of RaI function in cancers.

In this study, R428 was confirmed as a newly described orally bioavailable and selective small molecule antagonist of AxI function, (Holland et al., Cancer Res 2010; 70: 1544-54) that blocks invasion and anchorage independent growth of EAC cells in vitro. R428 inhibits in vivo metastases in multiple breast cancer preclinical models, which engenders the possibility that this agent might be beneficial in advanced EACs, especially in those adenocarcinomas that demonstrate evidence of AxI overexpression. AxI has also recently been implicated as a mechanism of chemoresi stance in solid tumors, particularly to ERBB2/Her-2new targeted therapies, such as lapatinib (Liu et al., Cancer Res 2009; 69:6871-8). Either pharmacological or genetic inhibition of AxI function abrogates phosphorylation of ERBB2 at the critical Tyr⁸⁷⁷ residue, and sensitizes OE33 cells to lapatinib and Iressa in vitro. Without wishing to be bound by theory, these results suggest there may be crosstalk between AxI and the ERBB/HER families, which might contribute to sustained receptor activation and resistance to targeted agents like lapatinib. Comparable cross-talk has been implicated between the epidermal growth factor receptor (EGFR) and insulin-like growth factor- 1 receptor (IGF-IR) pathways, rendering cancer cells resistant to EGFR-antagonist therapies. Therefore, combinatorial therapy using R428 or similar AxI inhibitors will likely provide additional efficacy in preclinical models of EAC in vivo.

The results reported herein were carried out using the following methods and materials. Sample collection for Serial Analysis of Gene Expression

SAGE libraries were constructed on endoscopic mucosal biopsies obtained from a 69 year old man with a diagnosis of Barrett esophagus, who progressed to EAC on surveillance. The histopathology on each of the four biopsies was confirmed on cryostat-embedded sections - using established criteria, and corresponded to normal squamous epithelium (NSE), low grade dysplasia in Barrett esophagus (LGD), high grade dysplasia in Barrett esophagus (HGD), and esophageal adenocarcinoma (EAC) (Figure 1). Total RNA was extracted using the MirVana RNA Isolation kit (Ambion, Austin, TX), as per the manufacturer's instructions. RNA integrity was confirmed on the Agilent 2100 Bioanalyzer, following which RNA quantification was performed on the Voctor2 (Perkin Elmer, Waltham, MA) coupled with RiboGreen dye

(Invitrogen, Carlsbad, CA).

Long SAGE library construction and data analysis

Four Long SAGE (L-SAGE) libraries were generated using lOμg of input RNA, with NIaIII as the anchoring enzyme and Mmel as the tagging enzyme, as previously described [⁴. The SAGE 2000 v4.5 software (http://www.sagenet.org) was used to extract SAGE tags, remove duplicate ditags, and tabulate tag counts. Linker sequences used in library construction, 1-bp sequence variations, and tag sequences occurring only once were removed from analysis. The SAGE tags were normalized to 200,000 tags, and statistical pair-wise comparison of tag frequencies in a binomial approach was performed as described by Romualdi et al. The esophagus L-SAGE library information and tag counts are posted at the Cancer Genome Anatomy Project's SAGE Genie web site (http://cgap.nci.nih.gov/SAGE). Unsupervised cluster analysis of genes and samples was performed using a subset of the most differentially expressed tags, using the TM4 software.

Barrett esophagus tissue microarrays (TMAs) for AxI and Gas6 immunohistochemistry Tissue microarrays (TMAs), representing 92 surgically resected EACs, and associated lesions from the histopathological spectrum of Barrett progression model, were used for AxI and Gas6 immunohistochemistry. Specifically, the non-neoplastic and precursor lesion "cores" on the TMAs included squamous epithelium (N=52), gastric cardiac epithelium (N = 70), non- dysplastic Barrett esophagus (N =15), LGD (N =13), and HGD (N =34); in addition, lymph node metastases were available for evaluation from 28 EAC cases. The construction and application of these TMAs for studying Barrett molecular progression have been previously described. [^{1> 2} Immunohistochemistry for AxI was performed using a polyclonal AxI primary antibody (catalog # AF154, R&D Systems, Minneapolis, MN, dilution 1 : 100), and for Gas6 using a polyclonal primary antibody (sc-1935, Santa Cruz Biotechnology, Santa Cruz, CA, dilution 1 : 100).

Labeling was detected with the Dako Universal LSAB™+ Kit/HRP (Dako, Inc. Carpinteria,

CA), as per the manufacturer protocol. The AxI receptor demonstrates mild cytoplasmic staining with a pronounced membrane accentuation, and only labeling of the appropriate cellular compartment was considered for evaluation. Intensity was scored on a three tier numerical scheme - 0 (absent), 1 (moderate), and 2 (intense), while area of staining was scored on a five tier numerical scheme - 0 (0%), 1 (<25%), 2 (26-50%), 3 (51-75%) and 4 (>75%); in cases with heterogeneous labeling intensity, the case was classified as per the predominant pattern of expression in the neoplastic cells. The two scores were multiplied in order to generate a composite "HistoScore", as previously described (Saha et al. Nat Biotechnol 2002; 20:508-12; Gjerdrum et al., Proc Natl Acad Sci U S A 2010; 107: 1 124-9). The lesions were classified as "absent/ low" AXL expressers (HistoScore of 0-3) or "high" AXL expressers (HistoScore of 4- 8). Reverse transcriptase and genomic Quantitative PCR (Q-PCR)

Reverse transcription was performed using Superscript II Reverse Transcriptase

(Invitrogen). Quantitative PCR was performed using either cDNA templates or genomic DNA templates on the ABI7300 Real Time PCR machine (Applied Biosystems, Foster City, CA), using SYBR®Green PCR Master Mix. Genomic Q-PCR for AXL gene dosage was performed by normalizing against a copy-number neutral internal control region (D0CK6) on chromosome 19p, and ratios relative to DNA copy number from JH-EsoAdl 's EBV-transformed B lymphocytes (germline DNA) for each AXL exon were calculated. Reverse transcriptase Q-PCR was performed using SDHA as the housekeeping gene. Each experiment was performed in triplicate. Primer sequences are available in Table 8.

Relative fold expression or exon dosage changes were calculated using the

method.

Genome-wide array CGH analysis of JH-EsoAdl

Copy number alterations in JH-EsoAdl genomic DNA were analyzed using Agilent 244K array CGH (Agilent Technologies, Santa Clara, CA). Briefly, genomic DNA was isolated using the PureGene DNA isolation kit (Gentra Systems, Minneapolis, MN), double-digested with Alul and Rsal (Promega, Madison, WI), labeled by random priming with either Cy3-dUTP (JH- EsoAdl) or Cy5-dUTP (Control), and hybridized according to the manufacturer's protocol. This oligonucleotide microarray contains 244,000 60-mer probes spanning coding and non-coding genomic sequences, with a median spacing of 7.4 and 16.5 kb respectively from NCBI Build 36 (March 2006) of the human genome. Arrays were analyzed using the Agilent DNA Microarray Scanner Model G2505B (5 micron resolution) and the Feature Extraction software (v9.1), followed by use of the Agilent CGH Analytics software (v.3.4). The array CGH data is in process of being submitted to the Gene Expression Omnibus (GEO) database, located at http://www.ncbi.nlin.nih.gov/gco/ Fluorescent in situ hybridization (FISH) for AXL gene amplification

The RPl 1-551E1O clone, spanning the 19ql3.1 region, was obtained from the Children's Hospital Oakland Research Institute. BAC DNA was isolated with the NucleoBond® BAC 100 (Clontech, Mountain View, CA). PCR products of AXL exons were resolved on an agarose gel to validate their genomic representation within the BAC. The DNA was labeled by

incorporation of SpectrumOrange labeled dUTPs using the Nick Translation Kit (Abbott, Abbott Park, IL). Labeled probes were precipitated in 100% ethanol, re-suspended in Hybridization Buffer (50% Formamide, 2xSSC, 10% Dextran Sulfate) and stored for further use at -80⁰C. Metaphase spreads were prepared from EBV-transformed B lymphocytes and JH-EsoAdl cells. Slides were pre-treated using Digest- All 3 (Invitrogen) at 1 : 100 dilution for 10 minutes at 37°C. Probes were applied on the slide, denatured for 5 minutes at 95°C, and then incubated at 37°C in a humid chamber. Slides were then washed in Wash buffer (2xSSC, 0.3 % NP-40) for 4 minutes at room temperature, 3 minutes at 75 ⁰C, counterstained with DAPI and mounted with Prolong Gold (Invitrogen). Images were captured using a Nikon E400 fluorescence microscope equipped with a Nikon DXM 1200 camera (Nikon Instruments, Melville, NY) and the SPOT Advanced digital imaging software (Diagnostic Instruments, Inc., SterlingHeights, MI).

Cell lines and culture conditions

We utilized NEK2, a spontaneously immortalized esophageal keratinocyte line,⁶ and

HEEPIC, which are non-immortalized primary esophageal keratinocytes,⁷ as control cells. Two Barrett-associated EAC cell lines were used: OE33 (European Collection of Cell Cultures, Wiltshire, UK) and JH-EsoAdl , as previously described (Linger et al., Adv Cancer Res 2008; 100:35-83, Zwick et al., Trends MoI Med 2002; 8: 17-23). The cells were maintained in RPMI- 1640 and supplemented with 10-20% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin.

Lentiviral shRNA knockdown

JH-EsoAdl and OE33 cells were seeded into 24-well plates at 9 x 10⁴ cells per well, and infected with either scrambled pLKO.l lentiviral vector (used as a "mock" in all experiments) or with lentivirus expressing AXL shRNA (Open Biosystems, Huntsville, AL). Stable clones were selected by adding 5 μg/ml of puromycin to the cell culture media. Reverse transcriptase Q-PCR analysis was used to select the best of 5 short hairpins for AXL mRNA knockdown {data not shown). All subsequent experiments were performed with AXL_sh4 (TRCNOOOOOO575, see Table 9), which achieved maximal knockdown of transcript.

Western blot analysis

Protein lysates were prepared with ImI ice-cold RIPA buffer (150 mM NaCl, 1.0% IGEP AL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0.) containing protease inhibitors (Complete Tablet, Roche, Nutley, NJ) and phosphatase inhibitor cocktail I&II (Sigma-Aldrich, St. Louis, MO). Proteins were separated by gel electrophoresis on NuPAGE Novex Bis-Tris 4-12% (Invitrogen) polyacrylamide gels and transferred to an Immobilon™-P PVDF Membrane (Sigma-Aldrich). Membranes were incubated with the following primary antibodies: anti-Axl (R&D Systems; dilution 1 : 1000), anti-phospho-Akt Ser⁴⁷³ (Cell Signaling Technology, Danvers, MA; dilution 1 : 1000), anti-phospho-p44/42 MAPK (Thr²⁰²/Tyr²⁰⁴) (Cell Signaling Technology; dilution 1 : 1000), anti-p44/42 MAPK (Erkl/2) (Cell Signaling

Technology; dilution 1 : 1000), anti-phospho-ERBB2 Tyr⁸⁷⁷ (Cell Signaling Technology; dilution 1 : 1000), anti-ERBB2 (Cell Signaling Technology; dilution 1 : 1000); anti-EGFR (Cell Signaling Technology; dilution 1 : 1000); anti-GAS6 (R&D Systems; dilution 1 :500); anti-actin (Santa Cruz Biotechnology; dilution 1 :5000); and anti-GAPDH (Fitzgerald Industries, Inc, Concord, MA; dilution: 1 :50,000). Signals were detected by ECL chemiluminescence (GE Healthcare, Piscataway, NJ). Signals were detected by ECL chemiluminescence (GE Healthcare,

Piscataway, NJ). RaI and Cdc42 activation assay

Levels of GTP-bound RaIA and RaIB proteins were determined using the RaI A&B Activation Assay kit (Upstate, Temecula, CA, USA), according to the manufacturer's instructions. OE33 cells were incubated with EGF (10ng/ml) for 10 min, prior to harvest. Using agarose beads bound to a GST fusion protein, GTP-bound RaI was precipitated, and levels of RaIA or RaIB quantified by Western blot analysis. Similarly, levels of active GTP-bound Cdc42 was determined using a Rho/Rac/Cdc42 Activation Assay Combo Kit (Cell Biolabs, Inc., San Diego, CA), following the standard protocol as recommended by the manufacturer Small molecule inhibition of AxI function

R428, an orally bioavailable small molecule inhibitor of AxI RTK, was discovered and synthesized at Rigel, Inc., and has been recently published (Holland et al., Cancer Res 2010; 70: 1544-54). In vitro cell viability assays

Cell viability assays using the The CellTiter 96®AQueousOne Solution Cell Proliferation Assay (Promega, Madison, WI) were performed on mock vector and AXL shRNA-expressing cells, as described previously ( Koorstra et al., Cancer Biol Ther 2009; 8:618-26). For viability studies in esophageal adenocarcinoma cells exposed to R428, gefitinib, lapatinib or the combination, cells were sub-cultured in a 96- well flat-bottomed plate, at 2000 cells / 50μl / well. After 24 hours, cells were exposed to varying concentration of compounds, for a period of 48 hours. Each treatment condition was assessed in triplicate wells, and each experiment was performed in three independent replicates at various times. At the end of exposure, 20 μL of MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium was added to each well, and the plates were incubated at 37 ⁰C for 1 hr. The inhibitory rate of cell proliferation was calculated by the following formula: Viability Rate (%) = [(ODtreated/ODcontrol)xl00%]. The IC₅₀ was calculated by using Hill three- parameter log fit. Modified Boyden chamber assays for migration and invasion

The modified Boyden chamber migration and invasion assays were performed as published previously (Koorstra et al., Cancer Biol Ther 2009; 8:618-26). For the invasion assay, a Matrigel™-coated 8-μm polypropylene filter inserts (BD Matrigel Matrix, BD Biosciences, Franklin Lakes, NJ) was used. All experiments were performed in triplicate.

Anchorage-independent growth in soft agar

Anchorage-independent growth was assessed by colony formation assays in soft-agar, as previously described (Koorstra et al., Cancer Biol Ther 2009; 8:618-26). Briefly, the soft agar assays were set up in 6-well plates, each well containing a bottom layer of 1% agarose

(Invitrogen), a middle layer of 0.7% agarose including 5 x 10³ cells, and a top layer of full medium only. The assay was terminated at day 14, when plates were stained with 0.005% crystal violet (Sigma-Aldrich) solution. Colony counting was performed for each triplicate condition using an automated ChemiDoc XRS instru-ment (Bio-Rad, Hercules, CA).

Heterotopic esophageal cancer xenografts.

Animal experiments were performed as per the guidelines of the Animal Care and Use Committee of Johns Hopkins University, and animals were maintained in accordance to standards of the American Association of Laboratory Animal Care. JH-EsoAdl cells (5 x 10⁶ in a total volume of 200 μL of 1/1 (v/v) PBS/Matrigel) were injected subcutaneously into the flank of male NOD/SCID mice (Charles River, Wilmington, MA), with one flank for the scrambled- transfected cells, and the other flank for the AXL shRNA-expressing cells (N = 3mice). The study was culminated after three weeks, at which time any visible xenografts were harvested and histology confirmed by H&E. No visible tumors were seen arising from the AXL shRNA expressing cells.

Statistical analysis.

Functional data are presented as mean and S. E. M., and were compared using a Student's t-test (or Mann-Whitney U-test, as appropriate). Survival analysis was performed using the Kaplan-Meier method and compared using the log-rank test. The proportional hazard regression analysis for predictors of survival was assessed by Cox's regression model. Significant values were defined as P<0.05 (SPSS, Chicago, IL). Graphs were illustrated using the GraphPad Prism 4.0.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A pharmaceutical composition for the treatment of a neoplasia comprising an effective amount of an agent that inhibits the expression or activity of AxI in combination with an effective amount of an agent that inhibits the expression or activity of EGF receptor and/or Her2.

2. The pharmaceutical composition of claim 1, wherein the agent that inhibits the expression or activity of AxI is a small compound, an anti-Axl antibody, or an AxI inhibitory nucleic acid molecule.

3. The pharmaceutical composition of claim 1, wherein the small compound is

R428.

4. The pharmaceutical composition of claim 1, wherein the agent that inhibits the expression or activity of EGF receptor is a small compound, an anti-EGF receptor antibody, or an EGF receptor inhibitory nucleic acid molecule.

5. The pharmaceutical composition of claim 4, wherein the agent is gefitinib (Iressa), Cetuximab (Erbitux), Erlotinib (Tarceva), Lapatinib, Panitumumab (Vectibix, ABX- EGF), RO5083945, IMC-11F8 (necitumumab), BMS-690514, BIBW2992 (Tovok), PF- 00299804, CUDC-101, or BIOMAb-EGFR (Nimotuzumab).

6. The pharmaceutical composition of claim 1, wherein the agent that inhibits the expression or activity of a Her2 receptor is a small compound, an anti-Her2 receptor antibody, or an Her2 inhibitory nucleic acid molecule.

7. The pharmaceutical composition of claim 6, wherein the agent is Trastuzumab (Herceptin^®), Lapatinib, or BIBW2992 (Tovok).

8. The pharmaceutical composition of claim 1, wherein the neoplasia is a cancer of epithelial, mesenchymal, hematological, or neural/neuroectodermal origin.

9. The pharmaceutical composition of claim 1, wherein the neoplasia is lung cancer, esophogeal carcinoma, colorectal carcinoma, breast cancer, pancreatic cancer, prostate cancer, cervical cancer, endometrial cancer, melanoma, glioblastoma, astrocytoma, or acute leukemia.

10. A method for treating or preventing a neoplasia, the method comprising administering to a subject an agent that inhibits the expression or activity of AxI in combination with an agent that inhibits the expression or activity of EGF receptor and/or Her2.

11. The method of claim 8, wherein the agent that inhibits the expression or activity of AxI is a small compound AxI inhibitor, an anti-Axl antibody, or an AxI inhibitory nucleic acid molecule.

12. The method of claim 8, wherein the small compound is R428 and derivatives thereof.

13. The method of claim 8, wherein the agent that inhibits the expression or activity of EGF receptor is a small compound, an anti-EGF receptor antibody, or an EGF receptor inhibitory nucleic acid molecule.

14. The method of claim 8, wherein the agent that inhibits the expression or activity of a Her2 receptor is a small compound, an anti-Her2 receptor antibody, or an Her2 inhibitory nucleic acid molecule.

15. The pharmaceutical composition of claim 1, wherein the neoplasia is a cancer of epithelial, mesenchymal, hematological, or neural/neuroectodermal origin.

16. A method of reducing the invasiveness of an esophageal adenocarcinoma cell in a subject, the method comprising contacting the neoplastic cell with an agent that inhibits AXL expression or biological activity in the cell relative to an untreated control cell.

17. A method of treating or preventing esophageal adenocarcinoma in a subject, the method comprising administering to the subject an agent that inhibits AXL expression or biological activity in the subject relative to a reference.

18. A method for inhibiting the progression of Barrett esophagus to esophageal adenocarcinoma in a subject, the method comprising administering to the subject an agent that inhibits inhibits AXL expression or biological activity in the cell relative to an untreated control cell.

19. A method for inhibiting the invasiveness of an esophageal adenocarcinoma in a subject, the method comprising administering to the subject an agent that inhibits AXL expression or biological activity in the cell relative to an untreated control cell.

20. The method of claim 16, wherein the cell is in vitro or in vivo.

21. The method of any one of claims 16-20, wherein the agent is an AXL inhibitory nucleic acid molecule selected from the group consisting of an antisense, siRNA or shRNA molecule.

22. The method of any one of claims 16-20, wherein the agent is a small compound that inhibits AxL receptor tyrosine kinase activity.

23. The method of claim 22, wherein the agent is R428.

24. The method of any one of claims 16-20, wherein the agent is an AXL -specific antibody.

25. The method of any one of claims 16-20, wherein the method further comprises administering to the subject an effective amount of lapatinib or Iressa.

26. The method of any one of claims 16-20, wherein the subject is a human.

27. The method of any one of claims 16-20, wherein the compound reduces neoplastic cell proliferation or invasiveness relative to the level in a corresponding neoplastic cell.

28. A pharmaceutical composition comprising an amount effective amount of one or more agents sufficient to inhibit the progression of Barrett esophagus to esophageal

adenocarcinoma in a subject, and a pharmaceutically-acceptable carrier.

29. A pharmaceutical composition comprising an amount effective amount of one or more agents sufficient to treat, prevent, or reduce the invasiveness of esophageal

adenocarcinoma in a subject, and a pharmaceutically-acceptable carrier.

30. The composition of claim 28 or 29, wherein the agent is R428.

31. The composition of claim 30, further comprising lapatinib.

32. A kit for the treatment of a neoplasia, the kit comprising the pharmaceutical composition of any of claims 28-30 and directions for using the kit for the treatment of a esophageal adenocarcinoma or for the prevention of the progression of Barrett esophagus to esophageal adenocarcinoma.

33. A method for characterizing Barrett esophagus in a subject, the method comprising comparing the expression or activity of an AxL polypeptide or polynucleotide in a biological sample of the subject to a reference level.

34. The method of claim 33, wherein the reference is the level of AxL present in a healthy control sample, a esophageal adenocarcinoma sample, or a sample from the same subject at an earlier point in time.

35. The method of claim 33, wherein the sample is a sample of esophageal epithelium or a lymph node sample.

36. The method of claim 33, wherein failure to detect or detection of minimal AxI expression indicates the presence of a non-dysplastic Barrett epithelium or a low-grade dysplasia.

37. The method of claim 33, wherein increased AxI expression indicates the presence of a high-grade dysplasia.

38. The method of claim 33, wherein an increased level of AxL in Barrett esophagus relative to a sample obtained at an earlier time point from the same subject indicates disease progression.

39. The method of claim 33, wherein AxL expression is measured in an

immunoassay, a radioassay, or other standard method.

40. A method for diagnosing a subject as having or having a propensity to develop esophageal adenocarcinoma, the method comprising detecting AxL expression or biological activity in a subject sample relative to a healthy control, thereby diagnosing esophageal adenocarcinoma in the subject.

41. A method for selecting an appropriate treatment for a subject having Barrett esophagus or esophageal adenocarcinoma, the method comprising measuring the expression or activity of AxL in a sample from the subject, wherein the level of AxL expression or activity indicates an appropriate treatment.

42. The method of claim 41, wherein minimal to largely absent AxI expression indicates that the subject should be monitored for progression of Barrett esophagus to esophageal adenocarcinoma.

43. The method of claim 41, wherein the subject is identified as in need of preventive therapy with an agent that inhibits the expression or activity of AxI.

44. The method of claim 41, wherein an increased level of AxI expression in an esophageal biopsy indicates that the subject is in need of aggressive treatment including multimodality chemotherapy with or without radiation to the cancer field.

45. The method of claim 41, wherein the subject is identified as in need of treatment with an agent that inhibits the expression or activity of AxI.

46. The method of claim 41 or 43, wherein the agent is R428.

47. The method of claim 46, wherein the treatment further comprises administering lapatinib to the subject.

48. The method of claim 41 or 43, wherein the sample comprises lymph node tissue.

49. The method of claim 48, wherein the identification of AxI expression or activity in lymph node tissue indicates that the subject has metastatic esophageal adenocarcinoma.

50. A method for monitoring the condition of a subject having Barrett's esophagus, the method comprising comparing the level of AxI expression or activity in a subject sample with the level present in a sample obtained at an earlier time, wherein a reduction in said AxI level identifies an improvement in the subject's condition, and an increase in said AxI level identifies a worsening in the subject's condition.

51. A method for determining the prognosis of a subject having Barrett' s esophagus or esophageal adenocarcinoma, the method comprising the level of AxI expression or activity in a subject sample with the level present in a reference, wherein the level of AxI in the sample is indicative of the subject's prognosis.

52. The method of claim 51, wherein an increased level of AxI indicates a poor prognosis and an increased level indicates a better prognosis.

53. A therapeutic composition for the cell- specific targeting of a therapeutic agent to an Axl-expressing cell, the composition comprising an anti-Axl antibody, aptamer, or other AxI binding agent linked to a chemotherapeutic agents, toxin or active fragments thereof, or radioactive isotope.

54. A diagnostic composition for the cell-specific targeting of a detectable agent to an Axl-expressing cell, the composition comprising an anti-Axl antibody, aptamer, or other AxI binding agent linked to a detectable moiety.