WO2010040083A2 - Prédicteurs de chimiorésistance par expression génique - Google Patents

Prédicteurs de chimiorésistance par expression génique Download PDF

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WO2010040083A2
WO2010040083A2 PCT/US2009/059423 US2009059423W WO2010040083A2 WO 2010040083 A2 WO2010040083 A2 WO 2010040083A2 US 2009059423 W US2009059423 W US 2009059423W WO 2010040083 A2 WO2010040083 A2 WO 2010040083A2
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myc
expression
egfr
nucleic acid
cancer
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PCT/US2009/059423
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WO2010040083A3 (fr
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Jeffrey E. Green
Hark K. Kim
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The U.S.A., As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2010040083A3 publication Critical patent/WO2010040083A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This disclosure relates to the field of cancer chemotherapy and in particular, to methods for predicting chemoresponsiveness in subjects with cancer, such as in subjects with epithelial cancer (e.g., gastric cancer), and for identifying treatment modalities for subjects with cancer.
  • epithelial cancer e.g., gastric cancer
  • BACKGROUND Gastric cancer is the second most common cause of cancer mortality worldwide. Many subjects with gastric cancer receive cytotoxic chemotherapy, but have a median survival of 9 to 11 months. Overall, cytotoxic chemotherapy prolongs the survival of metastatic gastric cancer patients, compared to the best supportive care, which historically results in an overall survival ranging from 3 to 5 months. Given the toxicity of chemotherapy, however, defining parameters that identify those subjects who will likely benefit from chemotherapy is of paramount importance.
  • Combination cisplatin and fluorouracil is a reference chemotherapy regimen for metastatic gastric cancer.
  • CF fluorouracil
  • Gene profiling signatures are disclosed herein that can be used to determine or predict a cancer subject's likely chemotherapy response, such as the response to cisplatin and fluorouracil chemotherapy in subjects with an epithelial cancer (such as gastric cancer, bladder cancer, head and neck cancer, esophageal cancer or cervical cancer).
  • an epithelial cancer such as gastric cancer, bladder cancer, head and neck cancer, esophageal cancer or cervical cancer.
  • the method of determining if a subject's tumor will be sensitive to treatment with a chemotherapeutic agent, such as CF includes detecting expression of chemotherapy sensitivity-related molecules including epithelial growth factor receptor (EGFR), fibroblast growth factor receptor type 2 (FGFR- 2) and MYC in a sample obtained from the subject.
  • EGFR epithelial growth factor receptor
  • FGFR- 2 fibroblast growth factor receptor type 2
  • MYC MYC
  • An increase in expression of these three molecules for example relative to a reference value representing expression of these three molecules in a subject that does not have cancer or has a chemosensitive cancer, indicates that the cancer has a decreased sensitivity to the chemotherapeutic agent (such as CF).
  • the subject may not respond to the chemotherapeutic agent in a manner sufficient to treat the cancer, and thus in some examples a decision may be made to not treat the subject with the chemotherapeutic agent (e.g., CF) or to treat the subject with a different therapy (e.g., a non-CF therapy).
  • the cancer is an epithelial-type cancer, including, but not limited to, gastric cancer, bladder cancer, head and neck cancer, esophageal cancer, or cervical cancer.
  • the method of determining whether a subject's tumor will be sensitive to treatment with a chemotherapeutic agent includes detecting expression of chemotherapy sensitivity-related molecules, including embryonic stem cell (ES) marker molecules and/or MYC target molecules, in a sample obtained from the subject.
  • ES embryonic stem cell
  • MYC target molecules such as one or more or even all of the molecules provided in Tables 10 or 7
  • An increase in expression of ES marker molecules and/or MYC target molecules indicates that the cancer has a decreased sensitivity (for example acquired or intrinsic chemoresistance) to the chemotherapeutic agent (such as CF).
  • the subject may not respond to the chemotherapeutic agent in a manner sufficient to treat the cancer, and thus in some examples a decision may be made to not treat the subject with the chemotherapeutic agent (e.g., CF) or to treat the subject with a different therapy (e.g., a non-CF therapy).
  • the chemotherapeutic agent e.g., CF
  • a different therapy e.g., a non-CF therapy
  • a different therapy e.g., a non-CF therapy
  • expression levels of ES marker molecules and/or MYC target molecules is similar to a reference value representing expression of these molecules in a subject that does not have cancer or has a chemosensitive cancer, this indicates that the cancer will be sensitive to the chemotherapeutic agent (such as CF), and thus in some examples a decision may be made to treat the subject with the chemotherapeutic agent (e.g., CF).
  • the cancer is an epithelial-type cancer, including, but not limited to
  • the method of determining whether a subject's tumor will be sensitive to treatment with a chemotherapeutic agent includes detecting tumor expression of chemotherapy sensitivity-related molecules, including EGFR, FGFR-2, MYC, and/or at least one ES molecule or MYC target molecule (such as one or more ES molecules provided in Table 10, such as one or more MYC target molecules provided in Table 7).
  • chemotherapy sensitivity-related molecules including EGFR, FGFR-2, MYC, and/or at least one ES molecule or MYC target molecule (such as one or more ES molecules provided in Table 10, such as one or more MYC target molecules provided in Table 7).
  • An increase in tumor expression of EGFR, FGFR-2, MYC, and/or one or more ES marker molecule or MYC target molecule, for example relative to a reference value representing expression of these molecules in a subject that does not have cancer or has a chemosensitive cancer indicates that the cancer has a decreased sensitivity (for example acquired or intrinsic chemoresistance) to the chemotherapeutic agent (such as CF).
  • the subject may not respond to the chemotherapeutic agent in a manner sufficient to treat the cancer, and thus in some examples a decision may be made to not treat the subject with the chemotherapeutic agent (e.g., CF) or to treat the subject with a different therapy (e.g. , a non-CF therapy).
  • the methods include detecting expression of the disclosed chemotherapy sensitivity -related molecules at either the nucleic acid level or protein level. In another example, the methods include determining whether a gene expression profile from the subject indicates chemoresponsiveness by using an array of molecules.
  • the array includes oligonucleotides complementary to at least molecules that encode EGFR, FGFR-2 and MYC. In another example, the array includes oligonucleotides complementary to at least one or more ES marker molecules or MYC target molecules (e.g. , one or more of the ES marker molecules provided in Table 10, such as one or more of the MYC target molecules provided in Table 7).
  • the array includes oligonucleotides complementary to at least molecules that encode EGFR, FGFR-2, MYC and one or more ES marker molecules or MYC target molecules (e.g. one or more of the ES marker molecules provided in Table 10, such as one or more of the MYC target molecules provided in Table 7).
  • the disclosed gene expression signatures have significant implications for the treatment of cancer.
  • the chemotherapy sensitivity-related molecules identified by the gene profile signatures can serve as targets for specific molecular therapeutic molecules that can increase the sensitivity of gastric cancer to standard chemotherapy.
  • the tumor has increased expression of EGFR, FGFR-2 and MYC
  • the subject can be administered inhibitors of EGFR, FGFR-2 and MYC biological activity to increase chemosensitivity of the tumor.
  • the tumor has increased expression of one or more ES marker molecules
  • the subject can be administered inhibitors of ES marker molecule biological activity to increase chemosensitivity of the tumor.
  • the subject can be administered inhibitors of MYC or MYC target molecule biological activity to increase chemosensitivity of the tumor.
  • the disclosed methods can further include administering to the subject a therapeutically effective treatment to increase sensitivity to the chemotherapeutic agent if the presence of differential expression indicates that the cancer, such as gastric cancer, has a decreased sensitivity to a chemotherapeutic agent.
  • the treatment includes administering a therapeutically effective amount of a composition, such as an agent that preferentially decreases biological activity of chemotherapy-sensitivity related molecules including EGFR, FGFR-2 and MYC.
  • the treatment includes administering a therapeutically effective amount of a composition, such as an agent that preferentially decreases biological activity of chemotherapy- sensitivity molecules including ES marker molecules (such as those provided in Table 10).
  • the treatment includes administering a therapeutically effective amount of a composition, such as an agent that preferentially decreases biological activity of chemotherapy- sensitivity molecules including MYC target molecules (such as those provided in Table 7).
  • the therapeutic agent can be an inhibitor of one or more of the chemotherapy-sensitivity related molecules, such as a siRNA or other RNAi molecules. Such inhibitors are useful for treatment of cancer, such as metastatic gastric cancer.
  • kits including arrays, antibodies or probes, for determining chemoresponsiveness of a cancer, such as an epithelial type cancer, including, but not limited to gastric cancer, bladder cancer, head and neck cancer, esophageal cancer or cervical cancer.
  • a cancer such as an epithelial type cancer, including, but not limited to gastric cancer, bladder cancer, head and neck cancer, esophageal cancer or cervical cancer.
  • the kit can include agents that can specifically detect EGFR, FGFR-2, and MYC nucleic acids or proteins.
  • the kit can include agents that can specifically detect ES marker nucleic acids or proteins.
  • the kit can include agents that can specifically detect MYC target nucleic acids or proteins.
  • Arrays can include other molecules, such as positive (including housekeeping genes) and negative controls as well as other chemotherapy-sensitivity related molecules, such as other cancer markers.
  • FIG. 2 provides Kaplan-Meier survival curves for two risk groups of the validation cohort predicted by the three-gene signature of MYC, EGFR and FGFR- 2.
  • the lines represent overall survival curves of predicted high- and low-risk groups, as indicated.
  • FIG. 3A is a Kaplan- Meier plot for time to progression calculated for each of the two major clusters generated using the 468 upregulated genes of the acquired resistance signature.
  • FIG. 3B is a Kaplan-Meier plot for overall survival of patients in the two clusters generated using the 468 upregulated genes of the acquired resistance signature.
  • FIG. 3C is a Kaplan- Meier plot for overall survival of 30 cisplatin-treated, advanced bladder cancer patients, clustered using the 468 upregulated genes of the acquired resistance signature.
  • FIG. 4A is a Venn diagram illustrating the selection of 50 MYC target genes in the upregulated genes of the acquired resistance signature.
  • 50 genes 60 probe sets were identified as MYC target genes.
  • FIG. 4B is a Kaplan-Meier plot for time to progression of patients clustered using the expression level of the 60 MYC target probe sets.
  • FIG. 4C is a Kaplan- Meier plot for overall survival of patients clustered using the expression level of the 60 MYC target probe sets. Patients in high-risk cluster (I) had a significantly shorter median survival than patients in low-risk cluster (II) (7.4 vs. 8.9 months, P ⁇ 0.006).
  • FIG. 5A is a Kaplan-Meier plot for proportion progression-free of 101 gastric cancer patients clustered using the 72-gene ES acquired resistance signature.
  • FIG. 5B is a Kaplan-Meier plot for overall survival of 101 gastric cancer patients clustered using the 72-gene ES acquired resistance signature.
  • FIG. 5C is a Kaplan- Meier plot for overall survival of 30 patients with cisplatin-treated advanced bladder cancer clustered using the 72-gene ES acquired resistance signature.
  • SEQ ID NOs: 1 and 2 are forward and reverse primers, respectively, used to amplify EGFR.
  • SEQ ID NO: 3 is an EGFR TaqMan probe.
  • SEQ ID NOs: 4 and 5 are forward and reverse primers, respectively, used to amplify FGFR-2.
  • SEQ ID NOs: 6 and 7 are forward and reverse primers, respectively, used to amplify MYC.
  • biomarkers for predicting a tumor's ⁇ e.g., epithelial cancer, such as gastric cancer
  • the inventors performed a prospective high-throughput gene expression study to identify transcriptional profiles that could predict a clinical response of metastatic gastric cancer subjects to chemotherapy.
  • gastric cancer was analyzed, the disclosed biomarkers are applicable to other epithelial cancers such as bladder cancer, head and neck cancer, esophageal cancer and cervical cancer.
  • Endoscopic biopsy samples were collected prior to chemotherapy from CF-treated metastatic gastric cancer subjects. High-throughput transcriptional profile data of biopsies from 96 training set subjects were correlated with the length of subject survival.
  • a survival risk predictor was constructed using the supervised principal component method, and used to predict the survival of 27 independent subjects in a validation cohort. These studies indicated that the over-expression of three genes in combination (EGFR, FGFR-2, and MYC) was highly predictive of poor response to chemotherapy. Over-expression of these three genes was confirmed at the protein level in tumors from subjects who had a poor response to chemotherapy, illustrating the clinical application of these three markers to improve the selection of therapeutic strategies for gastric cancer subjects and subjects with other types of epithelial tumors.
  • the inventors have identified a three gene expression profile that predicted overall survival of 27 independent patients after CF chemotherapy.
  • the adjusted hazard ratio of profile-defined poor-risk group was 3.15 (95% confidence interval, 1.18-8.38), and the significance was independent of age, performance status, and histologic type.
  • this three gene expression profile also predicted time to progression of 27 independent patients after CF chemotherapy. Time to progression, defined as time between the start of CF chemotherapy and the development of resistance, is a more specific measure of responsiveness to CF chemotherapy.
  • the p-value of three-gene profile prediction for time to progression was 0.027, and independent of age, performance status, and histologic type.
  • the inventors also identified a 468 gene expression profile that predicted acquired chemoresistance to CF therapy (tumor resistance to CF therapy in patients that initially responded to CF therapy).
  • This acquired chemoresistance gene signature included 72 genes that are known to be expressed in ES cells.
  • the acquired resistance signature also included 50 genes that are known to be regulated by MYC. These 50 MYC target genes were a subset of the 72-gene ES signature.
  • the acquired chemoresistance gene signature was also enriched in the gene expression signature identified in tumors with intrinsic chemoresistance to chemotherapy at presentation. Both the 72 ES marker genes and the 50 MYC target genes identified among the upregulated genes in the acquired chemoresistance signature were also predictive of response to chemotherapy.
  • the disclosed gene profiling signatures can be used to predict if a subject will be resistant or sensitive to standard chemotherapy, such as standard CF chemotherapy. This finding allows a subject's likely response to chemotherapy to be determined prior to receiving the treatment. For example, if expression of EGFR, FGFR-2 and MYC is increased, this indicates chemoresistance and in such a case a non-CF therapy may be selected and administered at a different dose (e.g., at an increased dose or frequency) or one or more agents that decrease the activity of EGFR, FGFR-2 and MYC may be administered prior to or at the time of CF therapy to increase chemosensitivity.
  • standard chemotherapy such as standard CF chemotherapy.
  • CF can be administered, as such a result indicates that the tumor is chemosensitive.
  • expression of one or more ES markers or MYC targets (such as the 72-gene ES marker profile or the 50-gene MYC target profile) is increased, this indicates chemoresistance, and in such a case a non-CF therapy may be selected and administered at a different dose (e.g. , at an increased dose or frequency) or one or more agents that decrease the activity of an ES marker molecule or a MYC target molecule may be administered prior to or at the time of CF therapy to increase chemosensitivity. If expression of one or more ES marker or MYC target is not increased, CF can be administered, as such a result indicates that the tumor is chemosensitive.
  • the method includes screening the subject to determine if they have an epithelial cancer, wherein subject's having an epithelial cancer (or have had such a cancer in the past), such as a gastric cancer, can be selected for chemosensitivity screening using the methods provided herein.
  • the disclosed gene signatures also identify genes and collections or sets of genes that serve as effective molecular markers for chemoresistance in cancer, such as epithelial cancer (e.g., gastric cancer or bladder cancer), as well as such genes or gene sets that can provide clinically effective therapeutic targets for cancer.
  • epithelial cancer e.g., gastric cancer or bladder cancer
  • methods are disclosed for increasing the sensitivity of a subject with epithelial cancer (such as a tumor that is chemoresistant, for example resistant to CF) to a chemotherapeutic agent by targeting the chemotherapy sensitivity-related molecules identified by the gene profile signatures.
  • a therapeutically effective amount of one or more agents e.g., 2 or more or 3 or more
  • agents can be siRNAs, antibodies, small inhibitory molecules, ap tamers and the like that inhibit such activity.
  • EGFR epidermal growth factor receptor ES: embryonic stem
  • FGFR-2 fibroblast growth factor receptor-2 HR: hazard ratios
  • nucleic acid molecule includes single or plural nucleic acid molecules and is considered equivalent to the phrase “comprising at least one nucleic acid molecule.”
  • or refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.
  • Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • injection such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous
  • oral sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Amplifying a nucleic acid molecule To increase the number of copies of a nucleic acid molecule, such as a gene or fragment of a gene, for example a region of a chemotherapy sensitivity-related gene, such as a gene that encodes EGFR, FGFR- 2, MYC, an ES marker, or a MYC target.
  • a nucleic acid molecule such as a gene or fragment of a gene, for example a region of a chemotherapy sensitivity-related gene, such as a gene that encodes EGFR, FGFR- 2, MYC, an ES marker, or a MYC target.
  • the resulting products are called amplification products.
  • PCR polymerase chain reaction
  • a biological sample obtained from a subject such as a sample containing cancer cells
  • a pair of oligonucleotide primers under conditions that allow for hybridization of the primers to a nucleic acid molecule in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid molecule.
  • in vitro amplification techniques include quantitative real-time PCR, reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), strand displacement amplification (see USPN 5,744,311); transcription-free isothermal amplification (see USPN 6,033,881); repair chain reaction amplification (see WO 90/01069); ligase chain reaction amplification (see EP-A-320 308); gap filling ligase chain reaction amplification (see USPN 5,427,930); coupled ligase detection and PCR (see USPN 6,027,889); and NASBATM RNA transcription-free amplification (see USPN 6,025,134).
  • RT-PCR reverse transcription PCR
  • qRT-PCR quantitative RT-PCR
  • strand displacement amplification see USPN 5,744,3111
  • transcription-free isothermal amplification see USPN 6,033,881
  • repair chain reaction amplification see WO 90/01069
  • ligase chain reaction amplification
  • a commonly used method for real-time quantitative polymerase chain reaction involves the use of a double stranded DNA dye (such as SYBR Green I dye). For example, as the amount of PCR product increases, more SYBR Green I dye binds to DNA, resulting in a steady increase in fluorescence.
  • Another commonly used method is real-time quantitative TaqMan PCR (Applied Biosystems). The 5' nuclease assay provides a real-time method for detecting only specific amplification products.
  • Antibody A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, such as an EGFR, FGFR2, MYC, ES marker, or MYC target protein or a fragment thereof.
  • Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
  • Fab' fragments fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv”).
  • the term also includes recombinant forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.
  • Array An arrangement of molecules, such as biological macromolecules
  • a "microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis. Arrays are sometimes called DNA chips or biochips.
  • the array of molecules makes it possible to carry out a very large number of analyses on a sample at one time. In certain example arrays, one or more molecules (such as an oligonucleotide probe) will occur on the array a plurality of times (such as twice), for instance to provide internal controls.
  • the number of addressable locations on the array can vary, for example from at least one, to at least 3, at least 10, at least 20, at least 30, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 500, least 550, at least 600, at least 800, at least 1000, at least 10,000, or more.
  • an array includes nucleic acid molecules, such as oligonucleotide sequences that are at least 15 nucleotides in length, such as about 15-40 nucleotides in length.
  • an array includes oligonucleotide probes or primers which can be used to detect sensitive to chemotherapy-associated sequences, such as nucleic acid sequences that encode EGFR, FGFR2, and MYC, either alone or with additional probes or primers to controls, including probes and primers to housekeeping genes (such as one or more of ⁇ -actin, glyceraldehyde 3 -phosphate dehydrogenase (GADPH), succinate dehydrogenase (SDHA), hypoxanthine phosphoribosyl transferase 1 (HRPTI), HBSl-like protein (HBSlL), a cyclophilin family member protein, and alpha haemoglobin stabilizing protein (AHSP)).
  • chemotherapy-associated sequences such as nucleic acid sequences that encode EGFR, FGFR2, and MYC
  • GADPH glyceraldehyde 3 -phosphate dehydrogenase
  • SDHA succinate dehydrogenase
  • an array includes oligonucleotide probes or primers which can be used to detect sensitive to chemotherapy-associated sequences, such as nucleic acids that encode one or more ES marker (such as those provided in Table 10) or MYC target (such as those provided in Table 7) either alone or with additional probes or primers to controls, including probes and primers to housekeeping genes.
  • the array is a commercially available array such as an oligonucleotide array from Affymetrix, Agilent, Nimbelgen, and Illumina.
  • each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array.
  • the feature application location on an array can assume different shapes.
  • the array can be regular (such as arranged in uniform rows and columns) or irregular.
  • the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position.
  • Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity).
  • the individual features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.
  • Protein-based arrays include probe molecules that are or include proteins, or where the target molecules are or include proteins, and arrays including nucleic acids to which proteins are bound, or vice versa.
  • an array contains antibodies to chemotherapy sensitivity-related proteins, such as EGFR, FGFR2, MYC, either alone or with additional antibodies such as to controls, such as 1 to 10 housekeeping proteins or other proteins (such as one or more of ⁇ -actin, glyceraldehyde 3-phosphate dehydrogenase (GADPH), succinate dehydrogenase (SDHA), hypoxanthine phosphoribosyl transferase 1 (HRPTI), HBSl -like protein (HBSlL), a cyclophilin family member protein, and alpha haemoglobin stabilizing protein (AHSP)).
  • the array contains antibodies to chemotherapy sensitivity-related proteins, such as ES marker proteins (for example, those provided in Table 10) or MYC target proteins (for example those provided in Table 7), either alone or with additional antibodies such as to controls.
  • Binding or stable binding An association between two substances or molecules, such as the hybridization of one nucleic acid molecule to another (or itself), the association of an antibody with a peptide, or the association of a protein with another protein or nucleic acid molecule.
  • An oligonucleotide molecule binds or stably binds to a target nucleic acid molecule if a sufficient amount of the oligonucleotide molecule forms base pairs or is hybridized to its target nucleic acid molecule (such as a nucleic acid molecule that encodes EGFR, FGFR2, MYC, an ES marker, or a MYC target), to permit detection of that binding.
  • an antibody binds or stably binds a target protein (e.g., EGFR, FGFR2, MYC, an ES marker, or a MYC target) when a sufficient amount of the antibody binds to its target protein (such as EGFR, FGFR2, MYC, an ES marker, or a MYC target), to permit detection of that binding.
  • a target protein e.g., EGFR, FGFR2, MYC, an ES marker, or a MYC target
  • a specific binding agent is an agent that binds substantially or preferentially only to a defined target such as a protein, enzyme, polysaccharide, oligonucleotide, DNA, RNA, recombinant vector or a small molecule.
  • a "specific binding agent” is capable of binding to at least one of the disclosed chemosensitivity molecules disclosed herein (such as EGFR, FGFR-2, MYC, ES markers or MYC targets). Binding can be detected by any procedure known to one skilled in the art, such as by physical or functional properties of the target:oligonucleotide complex or the target:protein complex (e.g., target: antibody complex). For example, binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, and the like or effect on biological activity of the nucleic acid or protein.
  • Physical methods of detecting the binding of complementary strands of nucleic acid molecules or binding of a target protein to an antibody include but are not limited to, such methods as DNase I or chemical footprinting, gel shift and affinity cleavage assays, in situ hybridization, Northern blotting, Western blotting, immunohistochemistry, dot blotting, and light absorption detection procedures.
  • one method involves observing a change in light absorption of a solution containing an oligonucleotide (or an analog) and a target nucleic acid at 220 to 300 nm as the temperature is slowly increased.
  • the method involves detecting a signal, such as a detectable label (e.g., fluorophore, chromogen, and the like), present on one or both nucleic acid molecules (or antibody or protein as appropriate).
  • a detectable label e.g., fluorophore, chromogen, and the like
  • T m The binding between an oligomer and its target nucleic acid is frequently characterized by the temperature (T m ) at which 50% of the oligomer is melted from its target.
  • T m the temperature at which 50% of the oligomer is melted from its target.
  • a higher (T m ) means a stronger or more stable complex relative to a complex with a lower (T m )-
  • Cancer A malignant tumor characterized by abnormal or uncontrolled cell growth. Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. "Metastatic disease” refers to cancer cells that have left the original tumor site and migrated to other parts of the body, for example via the bloodstream or lymph system.
  • cDNA complementary DNA: A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences which determine transcription. cDNA can be synthesized by reverse transcription from messenger RNA extracted from cells.
  • Chemoresistant or chemoresistance A condition (e.g., a tumor, such as an epithelial tumor, such as gastric cancer) that does not respond to an initial or subsequent chemotherapy treatment, such as CF chemotherapy.
  • a condition that does not respond to an initial chemotherapy treatment is referred to as having intrinsic chemoresistance.
  • a condition that responds to an initial chemotherapy treatment is referred to as having acquired chemoresistance.
  • Chemosensitive A condition (e.g., a tumor, such as an epithelial tumor, such as gastric cancer) that is significantly responsive to the initial (and in some examples subsequent) chemotherapy treatment.
  • chemosensitivity refers to the activity of any chemotherapeutic sensitivity-related molecule, including EGFR,
  • chemosensitivity refers to the activity of any chemotherapy sensitivity-related molecule, including one or more ES marker molecule (such as those provided in Table 10) or MYC target molecule (such as those provided in Table 7).
  • sensitivity refers to the activity of an agent (such as a chemotherapeutic agent) on the growth, development or progression of a disease, such as gastric cancer or bladder cancer.
  • a decreased chemosensitivity refers to a state in which a tumor is less responsive to a given chemotherapeutic agent as compared to a tumor that is responsive to the treatment.
  • sensitivity or responsiveness can be assessed using any endpoint indicating a benefit to the subject, including, without limitation, (1) inhibition, to some extent, of tumor growth, including slowing down and complete growth arrest; (2) reduction in the number of tumor cells; (3) reduction in tumor size or volume; (4) inhibition (such as reduction, slowing down or complete stopping) of tumor cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (such as reduction, slowing down or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but does not have to, result in the regression or rejection of the tumor; (7) relief, to some extent, of one or more symptoms associated with the tumor; (8) increase in the length of survival following treatment; and/or (9) decreased mortality at a given point of time following treatment.
  • Chemotherapeutic agent or Chemotherapy Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer.
  • a chemotherapeutic agent is an agent of use in treating cancer, such as epithelial cancer, including, but not limited to gastric cancer.
  • cancer such as epithelial cancer
  • One of skill in the art can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al , Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., 2000 Churchill Livingstone, Inc; Baltzer and Berkery.
  • chemotherapeutic agents used for treating epithelial cancer include carboplatin, cisplatin, capecitabine, paclitaxel, docetaxel, doxorubicin, epirubicin, topotecan, irinotecan, gemcitabine, etoposide, cyclophosphamide, methotrexate, fluorouracil and vinorelbine.
  • Combination chemotherapy is the administration of more than one agent to treat cancer.
  • a combination chemotherapy treatment that includes CF is administered to a subject with cancer, such as epithelial cancer ⁇ e.g., gastric cancer).
  • Chemotherapy sensitivity- related (or associated) molecule A molecule whose expression affects the ability of a tumor or cancer ⁇ e.g., epithelial cancer) to respond to chemotherapy ⁇ e.g., CF chemotherapy).
  • Such molecules include, for instance, EGFR, FGFR2, and MYC nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins, as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby) whose expression when upregulated decreases chemoresponsiveness.
  • Such molecules also include, for instance, ES marker nucleic acid sequences or MYC target nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins, as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby) whose expression when upregulated decreases chemoresponsiveness.
  • ES marker nucleic acid sequences or MYC target nucleic acid sequences such as DNA, cDNA, or mRNAs
  • proteins as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby) whose expression when upregulated decreases chemoresponsiveness.
  • Detection of expression of chemotherapy sensitivity -related molecules can be used to predict a subject's response to chemotherapy, such as to CF chemotherapy.
  • Chemotherapy sensitivity-related molecules can be involved in or influenced by cancer in different ways, including causative (in that a change in a chemotherapy sensitivity-related molecule leads to development of or progression of cancer that is chemoresistant) or resultive (in that development of or progression of cancer that is chemoresistant, causes or results in a change in the chemotherapy sensitivity-related molecule).
  • Complementarity and percentage complementarity Molecules with complementary nucleic acids form a stable duplex or triplex when the strands bind, (hybridize), to each other by forming Watson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when an oligonucleotide molecule remains detectably bound to a target nucleic acid sequence under the required conditions.
  • Complementarity is the degree to which bases in one nucleic acid strand base pair with the bases in a second nucleic acid strand. Complementarity is conveniently described by percentage, that is, the proportion of nucleotides that form base pairs between two strands or within a specific region or domain of two strands.
  • oligonucleotide For example, if 10 nucleotides of a 15-nucleotide oligonucleotide form base pairs with a targeted region of a DNA molecule, that oligonucleotide is said to have 66.67% complementarity to the region of DNA targeted.
  • sufficient complementarity means that a sufficient number of base pairs exist between an oligonucleotide molecule and a target nucleic acid sequence (such as a chemotherapy sensitivity-related molecule, for example EGFR, FGFR-2, MYC, ES markers, and MYC targets) to achieve detectable binding.
  • a target nucleic acid sequence such as a chemotherapy sensitivity-related molecule, for example EGFR, FGFR-2, MYC, ES markers, and MYC targets
  • the percentage complementarity that fulfills this goal can range from as little as about 50% complementarity to full (100%) complementary.
  • sufficient complementarity is at least about 50%, for example at least about 75% complementarity, at least about 90% complementarity, at least about 95% complementarity, at least about 98% complementarity, or even at least about 100% complementarity.
  • Contacting Placement in direct physical association, including both a solid and liquid form. Contacting can occur in vitro with isolated cells or tissue or in vivo by administering to a subject.
  • Determining expression of a gene product Detection of a level of expression in either a qualitative or quantitative manner, for example by detecting nucleic acid molecules or proteins by routine methods known in the art.
  • EGFR Epidermal Growth Factor Receptor
  • This protein is a receptor for members of the epidermal growth factor family.
  • EGFR is a cell surface protein that binds to epidermal growth factor. Binding of the protein to a ligand induces receptor dimerization and tyrosine autophosphorylation and leads to tumor cell proliferation, angiogenesis, invasion and metastasis, and inhibition of apoptosis. In particular examples, expression of EGFR is increased in chemoresistant gastric cancer.
  • GenBank Accession Nos.: NG_000726, NM_005228, NM_201284, NM_201283, and NM_201282 disclose EGFR nucleic acid sequences
  • GenBank Accession Nos.: AAB19486, AAH94761, AAI28420, and AAI18666 disclose EGFR protein sequences, all of which are incorporated by reference as provided by GenBank on September 15, 2008.
  • Epithelial cancer Any malignant neoplasm originating from epithelium, i.e. , a carcinoma.
  • exemplary epithelial cancers include gastric cancer, ovarian cancer, cervical cancer, bladder cancer, head and neck cancer, and esophageal cancer.
  • Expression The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein.
  • Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal.
  • Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.
  • nucleic acid molecule can be altered relative to a nucleic acid molecule, such as a normal (wild type) nucleic acid molecule.
  • Alterations in gene expression, such as differential expression include but are not limited to: (1) overexpression; (2) underexpression; or (3) suppression of expression.
  • Alternations in the expression of a nucleic acid molecule can be associated with, and in fact cause, a change in expression of the corresponding protein.
  • Protein expression can also be altered in some manner to be different from the expression of the protein in a normal (wild type) situation.
  • Controls or standards for comparison to a sample, for the determination of differential expression include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a tumor that is chemosensitive or from a subject not having a tumor) as well as laboratory values, even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.
  • Laboratory standards and values may be set based on a known or determined population value (e.g., a value representing expression of a gene for a particular parameter, such as epithelial cancer (e.g., gastric cancer) sensitive to chemotherapy or resistant to chemotherapy) and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.
  • a known or determined population value e.g., a value representing expression of a gene for a particular parameter, such as epithelial cancer (e.g., gastric cancer) sensitive to chemotherapy or resistant to chemotherapy
  • Fibroblast Growth Factor Receptor type 2 A member of the FGFR receptor tyrosine kinase family that can activate survival and antiapoptosis pathways through AKT and ERK kinases.
  • expression of FGFR-2 is increased in gastric cancer samples that are chemoresistant.
  • GenBank Accession Nos.: NM_023105-NM_023111, NM_022970 and NM_000141 disclose FGFR-2 nucleic acid sequences
  • GenBank Accession Nos.: ABL89213, CAA96492 and AAH39243 disclose FGFR-2 protein sequences, all of which are incorporated by reference as provided by GenBank on September 15, 2008.
  • Gastric cancer Cancer that forms in tissues lining the stomach. Treatment of gastric cancer depends mainly on the size and place of the tumor, the stage of disease, and the subject's general health. Treatment for gastric cancer can involve surgery, chemotherapy, or radiation therapy. Many subjects receive more than one type of treatment.
  • treatment for gastric cancer is determined by measuring the expression level of molecules that encode EGFR, FGFR-2, MYC, ES markers and/or MYC targets or EGFR, FGFR-2, MYC, ES marker, and/or MYC target proteins. If expression is not upregulated (as compared to a control or reference value, such as representing a chemosensitive tumor's level of expression or representing a normal non-cancer sample), then the gastric cancer is chemoresponsive and CF chemotherapy can be used to treat the cancer. If expression is upregulated compared to the control or reference vale, the gastric cancer is chemoresistant and an alternative therapy is used to treat the cancer.
  • Alternative treatments can include administering inhibitors of EGFR, FGFR-2, MYC, ES markers, and/or MYC targets prior to or concurrently with CF chemotherapy, to increase the sensitivity of the tumor to the chemotherapy.
  • Gene expression profile (or fingerprint): Differential or altered gene expression can be measured by changes in the detectable amount of gene expression (such as cDNA or mRNA) or by changes in the detectable amount of proteins expressed by those genes.
  • a distinct or identifiable pattern of gene expression for instance a pattern of high and low expression of a defined set of genes or gene- indicative nucleic acids such as ESTs; in some examples, as few as one or two genes provides a profile, but more genes can be used in a profile, for example at least 3, at least 4, at least 5, at least 6, at least 10, at least 20, at least 25, at least 30, at least 50, at least 80, at least 120 or more.
  • a gene expression profile (also referred to as a fingerprint) can be linked to a tissue or cell type (such as a gastric cancer cell), to a particular stage of normal tissue growth or disease progression (such as metastatic gastric cancer), or to any other distinct or identifiable condition that influences gene expression in a predictable way (e.g., chemoresistance and chemosensitive).
  • Gene expression profiles can include relative as well as absolute expression levels of specific genes, and can be viewed in the context of a test sample compared to a baseline or control sample profile (such as a sample from a subject who does not have cancer, such as gastric cancer, or has a chemosensitive cancer, such as chemosensitive gastric cancer).
  • a gene expression profile in a subject is read on an array (such as a nucleic acid or protein array).
  • an array such as a nucleic acid or protein array.
  • a gene expression profile is performed using a commercially available array such as an Affymetrix Human Genome U 133 Plus 2.0 Array
  • Hybridization To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na + concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold
  • Hybridization 5x SSC at 65°C for 16 hours Wash twice: 2x SSC at room temperature (RT) for 15 minutes each
  • Hybridization 5x-6x SSC at 65°C-70°C for 16-20 hours Wash twice: 2x SSC at RT for 5-20 minutes each
  • probes or primers to EGFR, FGFR-2, MYC, ES marker, and/or MYC target nucleic acids can be designed to EGFR, FGFR-2, MYC, ES marker, and/or MYC target sequences (such as to those sequences disclosed herein), thereby allowing such molecules to be detected in a sample, such as a tumor sample.
  • Inhibitor Any chemical compound, nucleic acid molecule, peptide such as an antibody, specific for a gene product that can reduce activity of a gene product or directly interfere with expression of a gene, such as genes that encode EGFR, FGFR-2, MYC, ES markers, and MYC targets that are upregulated in chemoresistant cancers, such as a chemoresistant epithelial cancer (e.g., a chemoresistant gastric cancer).
  • An inhibitor of the disclosure can inhibit the activity of a protein that is encoded by a gene either directly or indirectly. Direct inhibition can be accomplished, for example, by binding to a protein and thereby preventing the protein from binding an intended target, such as a receptor.
  • Indirect inhibition can be accomplished, for example, by binding to a protein's intended target, such as a receptor or binding partner, thereby blocking or reducing activity of the protein.
  • an inhibitor of the disclosure can inhibit a gene by reducing or inhibiting expression of the gene, inter alia by interfering with gene expression (transcription, processing, translation, post-translational modification), for example, by interfering with the gene's mRNA and blocking translation of the gene product or by post-translational modification of a gene product, or by causing changes in intracellular localization.
  • Isolated An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in the cell of the organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra- chromosomal DNA and RNA, proteins and cells.
  • Nucleic acid molecules and proteins that have been "isolated” include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.
  • an isolated cell can be a gastric cancer cell or other epithelial tumor cell that is substantially separated from other cell subtypes.
  • Label An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy.
  • a label can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein.
  • labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. ⁇ Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) and Ausubel et al. ⁇ In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).
  • MYC A proto-oncogene MYC of a transcription factor network that regulates cellular proliferation, replicative potential, growth, differentiation, and apoptosis.
  • expression of MYC is increased in gastric cancer samples that are chemoresistant.
  • GenBank Accession Nos.: NM_010849.4, NM_010849, NM_002467, and NP_034979.3 disclose MYC nucleic acid sequences
  • GenBank Accession Nos.: CAA46984, CAA25288, NP_002458, and AAB30748 disclose MYC protein sequences, all of which are incorporated by reference as provided by GenBank on September 15, 2008.
  • a "MYC target molecule” includes a nucleic acid or protein known to be regulated by MYC, either directly or indirectly.
  • a MYC regulated gene includes a gene that is activated (such as an increase in transcription or expression) or repressed (such as a decrease in transcription or expression) in the presence of MYC.
  • a MYC target molecule also includes a gene that includes one or more MYC-responsive element (for example, one or more MYC binding sites, such as an E box, for example, CACGTG) in its regulatory regions (for example, a nucleic acid 5' to a transcriptional start site, such as a promoter).
  • a MYC target molecule includes one or more of the molecules listed in Table 7.
  • Nucleic acid array An arrangement of nucleic acids (such as DNA or RNA) in assigned locations on a matrix, such as that found in cDNA arrays, or oligonucleotide arrays.
  • Nucleic acid molecules A deoxyribonucleotide or ribonucleotide polymer including, without limitation, cDNA, mRNA, genomic DNA, and synthetic (such as chemically synthesized) DNA.
  • the nucleic acid molecule can be double- stranded or single-stranded. Where single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. In addition, nucleic acid molecule can be circular or linear.
  • the disclosure includes isolated nucleic acid molecules having specified lengths of a chemotherapy sensitivity-related nucleotide sequence, such as EGFR, FGFR-2, MYC, ES markers, and MYC targets.
  • a chemotherapy sensitivity-related nucleotide sequence such as EGFR, FGFR-2, MYC, ES markers, and MYC targets.
  • Such molecules can include at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 50 consecutive nucleotides, such as 10-100 nucleotides of these publicly available sequences or more, and can be obtained from any region of a chemotherapy sensitivity-related molecule.
  • Oligonucleotide A plurality of joined nucleotides joined by native phosphodiester bonds, for example between about 6 and about 300 nucleotides in length.
  • An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions.
  • oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide.
  • oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a chemotherapy sensitivity-related molecule nucleic acid sequence (such as DNA or RNA) that is at least 6 nucleotides, for example at least 8, at least 10, at least 15, at least 20, at least 21, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100 or even at least 200 nucleotides long, or from about 6 to about 50 nucleotides, for example about 10-25 nucleotides, such as 12, 15 or 20 nucleotides of the publicly available EGFR, FGFR-2, MYC, ES marker, and MYC target sequences or more, can be obtained from any region of a chemotherapy sensitivity-related molecule.
  • a chemotherapy sensitivity-related molecule nucleic acid sequence such as DNA or RNA
  • a chemotherapy sensitivity-related molecule nucleic acid sequence such as DNA or RNA
  • oligonucleotide probe is a short sequence of nucleotides, such as at least 8, at least 10, at least 15, at least 20, at least 21, at least 25, or at least 30 nucleotides in length, used to detect the presence of a complementary sequence by molecular hybridization.
  • oligonucleotide probes include a label that permits detection of oligonucleotide probe: target sequence hybridization complexes.
  • Primers Short nucleic acid molecules, for instance DNA oligonucleotides 10-100 nucleotides in length, such as about 15, 20, 25, 30 or 50 nucleotides or more in length.
  • Primers can be annealed to a complementary target DNA strand (e.g., such as to EGFR, FGFR-2, MYC, ES marker, or MYC target) by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand.
  • Primer pairs can be used for amplification of a nucleic acid sequence, such as by PCR or other nucleic acid amplification methods known in the art.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • primers can be selected that include at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more consecutive nucleotides of a chemotherapy sensitivity-related nucleotide sequence.
  • Reference value An amount of activity or expression determined to be representative of a given condition. Reference values can include a range of values, real or relative expected to occur under certain conditions. These values can be compared with experimental values to determine if a given molecule is up-regulated or down-regulated in a particular sample for instance.
  • a reference value or range of values represents an amount of activity or expression of EGFR, FGFR-2, MYC, ES marker, and/or MYC target nucleic acid molecules or proteins in a chemosensitive tumor sample, such as a tumor that is sensitive to CF chemotherapy.
  • This value can then be used to identify tumors that are chemoresistant by comparing this reference value of EGFR, FGFR- 2, MYC, ES marker, and/or MYC target expression obtained from the test sample.
  • an increase in expression or activity of EGFR, FGFR-2 and MYC nucleic acid molecules or proteins in a tumor sample as compared to such a reference value indicates that the tumor is chemoresistant.
  • an increase in expression or activity of one or more ES marker or MYC target nucleic acid molecules or proteins (such as one or more ES markers provided in Table 10, for example, one or more MYC targets provided in Table 7) in a tumor sample as compared to such a reference value indicates that the tumor is chemoresistant.
  • a reference value or range of values represents an amount of activity or expression of EGFR, FGFR-2, MYC, ES marker and/or MYC target nucleic acid molecules or proteins in a chemoresistant tumor sample, such as a tumor that is resistant to CF chemotherapy.
  • This value can then be used to identify tumors that are chemoresistant by comparing this reference value of EGFR, FGFR- 2, MYC, ES marker, and/or MYC target expression obtained from the test sample.
  • this reference value EGFR, FGFR- 2, MYC, ES marker, and/or MYC target expression obtained from the test sample.
  • similar values in expression or activity of EGFR, FGFR-2 and MYC nucleic acid molecules or proteins in a tumor sample are observed as compared to such a reference value, this indicates that the tumor is chemoresistant.
  • Sample A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, serum, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material.
  • a sample includes a gastric cancer tissue biopsy, for example a metastatic gastric cancer tissue biopsy.
  • Sensitivity and specificity Statistical measurements of the performance of a binary classification test. Sensitivity measures the proportion of actual positives which are correctly identified (e.g., the percentage of tumors that are identified as being chemoresistant). Specificity measures the proportion of negatives which are correctly identified (e.g., the percentage of tumors identified as not being chemoresistant).
  • Short interfering RNA siRNA: A double stranded nucleic acid molecule capable of RNA interference or "RNAi.” (See, for example, Bass Nature 411: 428- 429, 2001; Elbashir et al, Nature 411: 494-498, 2001; and Kreutzer et al, International PCT Publication No.
  • siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides having RNAi capacity or activity.
  • a siRNA molecule is one that reduces or interferes with the biological activity of one or more chemotherapy sensitivity- related molecules including molecules that encode EGFR, FGFR-2, MYC, ES markers, or MYC targets.
  • Subject Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals, such as veterinary subjects.
  • Target sequence A sequence of nucleotides located in a particular region in the human genome that corresponds to a desired sequence, such as a chemotherapy sensitivity -related sequence.
  • the target can be for instance a coding sequence; it can also be the non-coding strand that corresponds to a coding sequence.
  • target sequences include those sequences associated with chemotherapy sensitivity, such as sequences that encode EGFR, FGFR-2, MYC, an ES marker, or a MYC target.
  • Therapeutically effective amount An amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response.
  • a therapeutic agent such as a chemotherapeutic agent, is administered in therapeutically effective amounts.
  • Effective amounts a therapeutic agent can be determined in many different ways, such as assaying for a reduction in tumor size or improvement of physiological condition of a subject having cancer, such as epithelial cancer (e.g., gastric cancer). Effective amounts also can be determined through various in vitro, in vivo or in situ assays.
  • cancer such as epithelial cancer (e.g., gastric cancer).
  • Effective amounts also can be determined through various in vitro, in vivo or in situ assays.
  • Therapeutic agents can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.
  • chemoresistance in the subject with cancer, such as gastric cancer.
  • Treatment can involve only slowing the progression to chemoresistance (for example resistance occurs after 6 months, such as 30 months from the initial chemotherapy treatment), but can also include halting or reversing chemoresistance permanently.
  • a pharmaceutical preparation can decrease chemoresistance by at least 20%, at least 50%, at least 70%, at least 90%, at least 98%, or even at least 100%, as compared to chemoresistance observed in the absence of the pharmaceutical preparation.
  • Treating a disease refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as a sign or symptom of gastric cancer. Treatment can also induce remission or cure of a condition, such as gastric cancer. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient.
  • Tumor All neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity.
  • a therapeutic agent to a gastric cancer cell or a subject sufficient to allow the desired activity.
  • the desired activity is decreasing the activity (such as the expression) of a chemotherapy sensitivity-related molecule (e.g., EGFR, FGFR-2, MYC, an ES marker, a MYC target or combinations of two or more thereof).
  • a chemotherapy sensitivity-related molecule e.g., EGFR, FGFR-2, MYC, an ES marker, a MYC target or combinations of two or more thereof.
  • Upregulated or activation When used in reference to the expression of a nucleic acid molecule, such as a gene, refers to any process which results in an increase in production of a gene product.
  • a gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein. Therefore, gene upregulation or activation includes processes that increase transcription of a gene or translation of mRNA.
  • Examples of processes that increase transcription include those that facilitate formation of a transcription initiation complex, those that increase transcription initiation rate, those that increase transcription elongation rate, those that increase processivity of transcription and those that relieve transcriptional repression (for example by blocking the binding of a transcriptional repressor).
  • Gene upregulation can include inhibition of repression as well as stimulation of expression above an existing level.
  • Examples of processes that increase translation include those that increase translational initiation, those that increase translational elongation and those that increase mRNA stability. Gene upregulation includes any detectable increase in the production of a gene product.
  • production of a gene product increases by at least 2-fold, for example at least 3-fold, at least 4-fold, at least 5-fold, at least 10- fold, or at least 20-fold as compared to a control (such an amount of gene expression in a normal cell or in a chemosensitive epithelial cancer cell, such as a chemosensitive gastric cancer cell).
  • a control is a relative amount of gene expression in a biological sample, such as in an epithelial tissue biopsy (e.g., gastric tissue biopsy) obtained from a subject that does not have cancer or has a cancer, such as an epithelial cancer (e.g., gastric cancer) that is chemosensitive.
  • chemotherapeutic response in subjects with cancer, such as subjects having or had an epithelial cancer (e.g., gastric cancer, bladder cancer, head and neck cancer, cervical cancer, or esophageal cancer).
  • epithelial cancer e.g., gastric cancer, bladder cancer, head and neck cancer, cervical cancer, or esophageal cancer.
  • chemoresistance such as intrinsic or acquired chemoresistance
  • chemosensitive such as CF chemotherapy.
  • CF chemotherapy includes treatment with cisplatin and fluorouracil and their equivalents, such as treatment with cisplatin and capecitabine.
  • the gene profiles and methods provided herein can predict with a sensitivity of at least 70% and a specificity of at least 80% for a chemoresistant cancer, such as a sensitivity of at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% (for example, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 83%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) and a specificity of at least of at least 80%, at least 85%, at least 90%, and at least 95% (for example, 81%, 82%, 83%, 84%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).
  • the gene expression profile includes molecules that encode at least EGFR, FGFR-2 and MYC that are indicative of chemoresistance.
  • the gene expression profile includes molecules that encode at least EGFR, FGFR-2 and MYC and other cancer markers, such as epithelial cancer markers (e.g., gastric cancer markers).
  • the gene expression profile includes molecules that encode one or more ES markers or MYC targets (such as one or more molecules provided in Tables 10 and 7, respectively) that are indicative of chemoresistance (such as acquired or intrinsic chemoresistance) or that encode at least one or more ES marker or MYC target and other cancer markers, such as epithelial cancer markers (e.g., gastric cancer markers).
  • Chemotherapy sensitivity-related molecules can include nucleic acid molecules (such as DNA, cDNA, or mRNAs) and proteins.
  • detecting expression of the chemotherapy sensitivity-related molecules includes detecting mRNA expression of the disclosed chemotherapy sensitivity-related molecules.
  • detecting expression of the chemotherapy sensitivity-related molecules includes detecting protein expression of the disclosed chemotherapy sensitivity -related molecules.
  • an increase in the expression or biological activity of one or more of the disclosed chemotherapy sensitivity-related molecules includes an increase in production of a gene product, such as RNA or protein.
  • an increase in the expression or biological activity of one or more of the disclosed chemotherapy sensitivity-related molecules includes an increase in production of a gene product, such as RNA or protein.
  • a gene product such as RNA or protein.
  • processes that increase transcription of a gene or translation of mRNA are included.
  • Gene upregulation includes any detectable increase in the production of a gene product.
  • production/expression of a gene product is considered to be increased if the amount of chemotherapy sensitivity-related nucleic acid or protein detected in the test sample is increased by at least 2-fold, for example at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold as compared to a control (such as an amount of gene or protein expression in a normal cell, such as a normal epithelial cell (e.g., normal gastric epithelial cell), or a cancer cell that is chemosensitive, such as a chemosensitive epithelial cancer cell). If such upregulation is detected, this indicates that the subject has a chemoresistant epithelial (e.g., gastric) tumor.
  • a chemoresistant epithelial e.g., gastric
  • genes with a positive t-value such as molecules that encode at least EGFR, FGFR-2 and MYC
  • chemoresistant epithelial cancers e.g., gastric cancer
  • genes with a positive t-value such as molecules that encode one or more ES marker or MYC target (such as one or more gene provided in Tables 10 and 7, respectively) are upregulated in chemoresistant (for example, acquired or intrinsic chemoresistant) epithelial cancers (such as gastric cancer) relative to chemosensitive cancers.
  • genes with a positive t- value such as molecules that encode at least EGFR, FGFR-2, MYC, and one or more ES marker or MYC target (such as one or more MYC targets provided in Table 7 or one or more ES markers as provided in Table 10) are upregulated in chemoresistant epithelial cancers (e.g. gastric cancer) relative to chemosensitive cancers.
  • chemoresistant epithelial cancers e.g. gastric cancer
  • a control is a relative amount of gene or protein expression in a biological sample, such as in a tissue biopsy (e.g., epithelial sample) obtained from a subject that does not have cancer or a tumor biopsy (e.g., epithelial cancer sample) obtained from a subject that has a chemosensitive epithelial cancer.
  • a control is relative to a standard or reference value of the gene expression or protein expression expected to be present in a subject who does not have cancer or from a subject that has a chemosensitive cancer. Reference values can include a range of values, real or relative expected to occur under certain conditions. These values can be compared with experimental values to determine if a given molecule is up-regulated or down-regulated.
  • Methods are disclosed herein for determining if a subject has a tumor (or for example had a surgically resected tumor) that is sensitive to treatment with a chemotherapeutic agent, such as combination cisplatin and fluorouracil chemotherapy.
  • Subjects can be screened to determine whether the subject with a tumor, such as epithelial cancer (e.g., gastric cancer), is or will become chemoresistant by using the disclosed gene signature profiles.
  • a tumor such as epithelial cancer (e.g., gastric cancer)
  • increased expression of at least EGFR, FGFR-2 and MYC such as an increase of at least 2-, 3-, 4-, 5-, 10-, or 20-fold
  • a control/reference value representing chemosensitivity can indicate that the subject is likely not to respond to standard chemotherapy, such as CF chemotherapy.
  • increased expression of one or more ES markers (such as an increase of at least 2-, 3-, 4-, 5-, 10-, or 20- fold of one or more ES markers provided in Table 10 or all of the markers in Table 10) relative to a control/reference value representing chemosensitivity can indicate that the subject is likely not to respond to standard chemotherapy, such as CF chemotherapy (intrinsic chemoresistance) or is likely not to respond to subsequent treatment with the chemotherapy (acquired chemoresistance).
  • standard chemotherapy such as CF chemotherapy (intrinsic chemoresistance) or is likely not to respond to subsequent treatment with the chemotherapy (acquired chemoresistance).
  • increased expression of one or more MYC targets can indicate that the subject is likely not to respond to standard chemotherapy, such as CF chemotherapy (intrinsic chemoresistance) or is likely not to respond to subsequent treatment with the chemotherapy (acquired chemoresistance).
  • standard chemotherapy such as CF chemotherapy (intrinsic chemoresistance) or is likely not to respond to subsequent treatment with the chemotherapy (acquired chemoresistance).
  • the methods can be used to determine if the subject is a candidate for receiving standard chemotherapies or one of the therapies disclosed herein.
  • the chemotherapy sensitivity-related molecules are detected in a biological sample.
  • the biological sample is a tumor biopsy, such as an epithelial tumor biopsy (such as a gastric tumor biopsy).
  • chemotherapy sensitivity-related molecules are detected in a blood or urine sample, such as chemotherapy sensitivity-related molecules secreted or cell surface molecules that are susceptible to enzymatic cleavage at the cell surface.
  • chemoresponsiveness can be screened for by detecting at least EGFR, FGFR-2 and MYC.
  • the method can include detecting at least EGFR, FGFR-2 and MYC as well as other chemosensitivity-related molecules.
  • Such molecules include, for instance, nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins. Specific genes include EGFR, FGFR-2 and MYC as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby).
  • chemoresponsiveness can be screened for by detecting one or more ES cell markers.
  • the method can include detecting one or more ES markers (such as one or more ES markers provided in Table 10 or all of the markers in Table 10) as well as other chemosensitivity-related molecules.
  • Such molecules include, for instance, nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins. Specific genes include ES markers provided in Table 10, as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby).
  • chemoresponsiveness can be screened for by detecting one or more MYC targets.
  • the method can include detecting one or more MYC targets (such as one or more MYC targets provided in Table 7 or all of the markers in Table 7) as well as other chemosensitivity-related molecules.
  • MYC targets such as one or more MYC targets provided in Table 7 or all of the markers in Table 7.
  • Such molecules include, for instance, nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins.
  • Specific genes include MYC targets provided in Table 7, as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby).
  • the method indicates if a subject is or is likely to become chemoresistant.
  • increased or over-expression of at least EGFR, FGFR-2 and MYC is indicative of chemoresistance.
  • the method can identify an epithelial tumor (e.g., gastric tumor) as chemoresistant by measuring expression of EGFR, FGFR-2 and MYC, wherein increased expression in these three molecules (e.g., relative to a normal sample or a chemosensitive epithelial tumor sample) indicates the tumor is chemoresistant.
  • increased or over-expression of one or more ES marker targets (such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 72 ES markers, for example, one or more ES markers provided in Table 10) is indicative of chemoresistance (such as acquired or intrinsic chemoresistance).
  • increased or over-expression of one or more MYC targets (such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 MYC targets, for example, one or more MYC targets provided in Table 7) is indicative of chemoresistance (such as acquired or intrinsic chemoresistance).
  • the method can identify an epithelial tumor (such as gastric tumor) as chemoresistant by measuring expression of one or more ES markers, wherein increased expression (e.g., relative to a normal sample or a chemosensitive epithelial tumor sample) of the one or more ES markers indicates the tumor is or is likely to become chemoresistant.
  • the method can identify an epithelial tumor (such as gastric tumor) as chemoresistant by measuring expression of one or more MYC targets, wherein increased expression
  • the method can include detecting expression of chemotherapy sensitivity- related molecules at either the nucleic acid level or protein level. Certain methods involve determining whether a gene expression profile from the subject indicates chemoresponsiveness by using an array of molecules.
  • the array can include, consist essentially of, or consist of oligonucleotides complementary to EGFR, FGFR-2 and MYC.
  • the array includes, consists essentially of, or consists of oligonucleotides complementary to at least EGFR, FGFR-2 and MYC and one or more positive or negative controls (e.g., housekeeping genes such as ⁇ -actin and GADPH).
  • the array can include, consist essentially of, or consist of oligonucleotides complementary to one or more ES makers or MYC targets (such as one or more ES markers provided in Table 10, for example, one or more MYC targets provided in Table 7).
  • the array can include, consist essentially of, or consist of oligonucleotides complementary to one or more ES markers or MYC targets (such as one or more ES markers provided in Table 10, for example, one or more MYC targets provided in Table 7) and one or more positive or negative controls (e.g., housekeeping genes such as ⁇ -actin and GADPH).
  • the array can include oligonucleotides complementary to at least EGFR, FGFR-2, MYC and one or more ES markers (such as one or more ES markers provided in Table 10) and/or one or more MYC targets (such as one or more MYC targets provided in Table 7).
  • the array can include oligonucleotides complementary to at least EGFR, FGFR-2, MYC, and one or more ES markers (such as one or more ES markers provided in Table 10) and one or more positive or negative controls (e.g., housekeeping genes such as ⁇ -actin and GADPH).
  • the array can include oligonucleotides complementary to at least EGFR, FGFR-2, MYC, and one or more MYC targets (such as one or more MYC targets provided in Table 7) and one or more positive or negative controls (e.g., housekeeping genes such as ⁇ -actin and GADPH).
  • MYC targets such as one or more MYC targets provided in Table 7
  • positive or negative controls e.g., housekeeping genes such as ⁇ -actin and GADPH.
  • the disclosed arrays can include other molecules such as other cancer markers, such as other epithelial cancer markers (e.g. , gastric cancer markers).
  • nucleic acids in a biological sample are isolated, amplified, or both, prior to detecting expression.
  • amplification and detection of expression occur simultaneously or nearly simultaneously.
  • nucleic acid expression can be detected by PCR (for example, RT-PCR or quantitative RT-PCR).
  • PCR for example, RT-PCR or quantitative RT-PCR.
  • nucleic acids can be isolated and amplified by employing commercially available kits.
  • the biological sample can be incubated with primers that permit the amplification of at least EGFR, FGFR-2 and MYC mRNAs, under conditions sufficient to permit amplification of such products.
  • the biological sample can be incubated with primers that permit the amplification of one or more ES markers or MYC targets (such as one or more ES markers provided in Table 10, for example, one or more MYC targets provided in Table 7).
  • primers that permit the amplification of one or more ES markers or MYC targets (such as one or more ES markers provided in Table 10, for example, one or more MYC targets provided in Table 7).
  • the resulting amplicons can be detected.
  • the biological sample is incubated with probes that can bind to at least EGFR, FGFR-2 and MYC nucleic acid molecules (such as cDNA, genomic DNA, or RNA (such as mRNA)) under high stringency conditions.
  • the biological sample is incubated with probes that can bind to one or more ES marker or MYC target (such as one or more ES markers provided in Table 10, for example, one or more MYC targets provided in Table 7) nucleic acid molecules (such as cDNA, genomic DNA, or RNA (such as mRNA)) under high stringency conditions.
  • the resulting hybridization can then be detected using methods known in the art.
  • a subject is screened by applying isolated nucleic acid molecules obtained from a biological sample including cancer cells, such as epithelial cancer cells, including gastric cancer cells, to an array.
  • the array includes oligonucleotides complementary to at least molecules that encode EGFR, FGFR-2 and MYC.
  • the array includes oligonucleotides complementary to at least one or more ES markers (such as one or more ES markers provided in Table 10).
  • the array includes oligonucleotides complementary to at least one or more MYC targets (such as one or more MYC targets provided in Table 7).
  • the array is a commercially available array such as an Affymetrix Human Genome U133 Plus 2.0 Array.
  • the isolated nucleic acid molecules are incubated with the array including oligonucleotides complementary to at least molecules that encode EGFR, FGFR-2 and MYC for a time sufficient to allow hybridization between the isolated nucleic acid molecules and oligonucleotide probes, thereby forming isolated nucleic acid molecule: oligonucleotide complexes.
  • the isolated nucleic acid molecule: oligonucleotide complexes are then analyzed to determine if expression of the isolated nucleic acid molecules is increased.
  • Increased expression in molecules that encode EGFR, FGFR-2 and MYC indicates that the cancer cells have a decreased sensitivity to a chemotherapeutic agent, such as CF.
  • the isolated nucleic acid molecules are incubated with the array including oligonucleotides complementary to at least one or more ES marker or MYC targets (such as one or more ES markers provided in Table 10, for example, one or more MYC targets provided in Table 7) for a time sufficient to allow hybridization between the isolated nucleic acid molecules and oligonucleotide probes, thereby forming isolated nucleic acid molecule:oligonucleotide complexes.
  • the isolated nucleic acid molecule:oligonucleotide complexes are then analyzed to determine if expression of the isolated nucleic acid molecules is increased.
  • Increased expression in molecules that encode one or more ES marker or MYC target indicates that the cancer cells have or are likely to develop a decreased sensitivity to a chemotherapeutic agent, such as CF.
  • proteins can be detected using routine methods such as Western blot, immunohistochemistry, or mass spectrometry. In some examples, proteins are purified before detection.
  • chemotherapy sensitivity-related proteins such as EGFR, FGFR-2 and MYC, can be detected by incubating the biological sample (such as a cancer sample) with an antibody that specifically binds to one or more of the disclosed chemotherapy sensitivity-related proteins.
  • chemotherapy sensitivity-related proteins such as one or more ES markers or MYC targets (such as one or more ES markers provided in Table 10, for example, one or more MYC targets provided in Table 7) can be detected by incubating the biological sample (such as a cancer sample) with an antibody that specifically binds to one or more of the disclosed chemotherapy sensitivity-related proteins.
  • the primary antibody can include a detectable label.
  • the primary antibody can be directly labeled, or the sample can be subsequently incubated with a secondary antibody that is labeled (for example with a fluorescent label).
  • the label can then be detected, for example by microscopy, ELISA, flow cytometery, or spectrophotometry.
  • the biological sample is analyzed by Western blotting for detecting expression of EGFR, FGFR-2 and MYC.
  • the biological sample is analyzed by Western blotting for detecting expression of one or more ES marker or MYC target.
  • the antibody that specifically binds a chemotherapy sensitivity-related molecule (such as EGFR, FGFR-2 and MYC, one or more ES marker or one or more MYC target) is directly labeled with a detectable label.
  • each antibody that specifically binds a chemotherapy sensitivity- related molecule (the first antibody) is unlabeled and a second antibody or other molecule that can bind the human antibody that specifically binds the respective chemotherapy sensitivity-related molecule is labeled.
  • a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody.
  • the secondary antibody can be an anti-human-IgG.
  • Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
  • Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin.
  • Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • a non-limiting exemplary luminescent material is luminol; a non- limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 1, 131 1, 35 S or 3 H.
  • chemotherapy sensitivity-related molecules can be assayed in a biological sample by a competition immunoassay utilizing chemotherapy sensitivity-related molecule standards labeled with a detectable substance and an unlabeled antibody that specifically binds the desired chemotherapy sensitivity-related molecule.
  • the biological sample such as tissue biopsy, cells isolated from a tissue biopsy, serum, or urine
  • the labeled chemotherapy sensitivity-related molecule standards and the antibody that specifically binds the desired chemotherapy sensitivity-related molecule are combined and the amount of labeled chemotherapy sensitivity-related molecule standard bound to the unlabeled antibody is determined.
  • the amount of chemotherapy sensitivity-related molecule in the biological sample is inversely proportional to the amount of labeled chemotherapy sensitivity-related molecule standard bound to the antibody that specifically binds the chemotherapy sensitivity- related molecule.
  • a subject is screened by determining whether they have increased expression (such as an increase of at least 2-fold, 5-fold, 10-fold, or 20- fold) of one or more of the disclosed chemotherapy sensitivity-related proteins, including at least EGFR, FGFR-2 and MYC, relative to a normal sample or a chemosensitive epithelial cancer sample.
  • increased expression such as an increase of at least 2-fold, 5-fold, 10-fold, or 20- fold
  • the disclosed chemotherapy sensitivity-related proteins including at least EGFR, FGFR-2 and MYC
  • a subject is screened to determine whether they have increased levels of EGFR, FGFR-2 and MYC in a sample, such as a tissue sample, for example a gastric tumor sample (for example relative to a level present in a sample from a subject not having a tumor or having a chemosensitive epithelial cancer, such as a chemosensitive gastric cancer), for example using an antibody that specifically binds one or more of the disclosed chemotherapy sensitivity-related molecule (such as those described below).
  • a sample such as a tissue sample, for example a gastric tumor sample (for example relative to a level present in a sample from a subject not having a tumor or having a chemosensitive epithelial cancer, such as a chemosensitive gastric cancer), for example using an antibody that specifically binds one or more of the disclosed chemotherapy sensitivity-related molecule (such as those described below).
  • a subject is screened by determining whether they have increased expression (such as an increase of at least 2-fold 5-fold, 10-fold, or 20- fold) of one or more of the disclosed chemotherapy sensitivity-related proteins, including one or more ES markers (such as those provided in Table 10) or MYC targets (such as those provided in Table 7), relative to a normal sample or a chemosensitive epithelial cancer sample.
  • increased expression such as an increase of at least 2-fold 5-fold, 10-fold, or 20- fold
  • the disclosed chemotherapy sensitivity-related proteins including one or more ES markers (such as those provided in Table 10) or MYC targets (such as those provided in Table 7)
  • a subject is screened to determine whether they have increased levels of one or more ES markers or MYC targets in a sample, such as a tissue sample, for example a gastric tumor sample (for example relative to a level present in a sample from a subject not having a tumor or having a chemosensitive epithelial cancer, such as a chemosensitive gastric cancer), for example using an antibody that specifically binds one or more of the disclosed chemotherapy sensitivity -related molecules (such as those described herein).
  • a sample such as a tissue sample, for example a gastric tumor sample (for example relative to a level present in a sample from a subject not having a tumor or having a chemosensitive epithelial cancer, such as a chemosensitive gastric cancer)
  • an antibody that specifically binds one or more of the disclosed chemotherapy sensitivity -related molecules (such as those described herein).
  • the expression of chemotherapy- sensitivity related molecules can be compared to a reference value or control sample to determine if there is differential expression of the detected molecules.
  • the expression of chemotherapy-sensitivity related molecules including EGFR, FGFR-2 and MYC detected in a test sample is compared to a reference value, such as an amount of the given gene or protein expected to be expressed in a cancer cell, such as an epithelial cancer cell (e.g., gastric cancer cell), obtained from a subject who does not have cancer, such as gastric cancer or who has gastric cancer that is chemoresponsive.
  • the expression level of EGFR, FGFR-2 and MYC is compared to a control sample, such as a sample obtained from a subject who does not have cancer, such as epithelial cancer (e.g., gastric cancer), or who has a chemoresponsive cancer, such as a chemoresponsive epithelial cancer (e.g., gastric cancer).
  • a control sample such as a sample obtained from a subject who does not have cancer, such as epithelial cancer (e.g., gastric cancer), or who has a chemoresponsive cancer, such as a chemoresponsive epithelial cancer (e.g., gastric cancer).
  • the expression of chemotherapy- sensitivity related molecules including one or more ES markers or MYC targets (such as one or more ES markers provided in Table 10 or one or more MYC targets provided in Table 7) detected in a test sample is compared to a reference value, such as an amount of the given gene or protein expected to be expressed in a cancer cell, such as an epithelial cancer cell (e.g., gastric cancer cell), obtained from a subject who does not have cancer, such as gastric cancer or who has gastric cancer that is chemoresponsive.
  • a cancer cell such as an epithelial cancer cell (e.g., gastric cancer cell)
  • an epithelial cancer cell e.g., gastric cancer cell
  • the expression level of one or more ES markers or MYC targets is compared to a control sample, such as a sample obtained from a subject who does not have cancer, such as epithelial cancer (e.g., gastric cancer), or who has a chemoresponsive cancer, such as a chemoresponsive epithelial cancer (e.g. , gastric cancer).
  • a control sample such as a sample obtained from a subject who does not have cancer, such as epithelial cancer (e.g., gastric cancer), or who has a chemoresponsive cancer, such as a chemoresponsive epithelial cancer (e.g. , gastric cancer).
  • chemotherapy sensitivity is associated with differential expression of chemotherapy sensitivity-related molecules.
  • a disclosed gene expression profile has identified three chemotherapy sensitivity- related molecules, EGFR, FGFR-2 and MYC, associated with chemoresistance.
  • An additional disclosed gene expression profile has identified 72 chemotherapy sensitivity related molecules which are ES cell markers and 50 chemotherapy sensitivity-related molecules, which are MYC targets, associated with chemoresistance (such as acquired or intrinsic chemoresistance).
  • methods of treatment to alter sensitivity to a chemotherapeutic agent such as chemoresistance associated with cancer, including epithelial cancer (e.g., gastric cancer).
  • the method includes determining if the subject has a tumor, such as a metastatic gastric tumor, that is chemoresistant (e.g., any methods provided herein). If negative, the subject is chemosensitive and standard chemotherapy can be administered. If the subject has a tumor, such as an epithelial tumor (e.g., gastric tumor), that is chemoresistant, then agents can be administered to reverse the pattern of expression of the genes/proteins associated with the chemoresistance.
  • a tumor such as a metastatic gastric tumor, that is chemoresistant
  • an epithelial tumor e.g., gastric tumor
  • agents can be administered to decrease the expression of nucleic acids that encode at least EGFR, FGFR-2 and MYC or decrease expression of EGFR, FGFR-2 and MYC proteins themselves.
  • agents can be administered to decrease the expression of nucleic acids that encode at least one or more ES markers or MYC targets (such as those provided in Tables 10 and 7, respectively) or decrease expression of at least one ES marker or MYC target (such as those provided in
  • a therapy for treating chemoresistance e.g., increasing chemosensitivity of the tumor by at least 20%, at least 50%, or at least 75%, relative to for example the absence of therapy
  • chemoresistance e.g., increasing chemosensitivity of the tumor by at least 20%, at least 50%, or at least 75%, relative to for example the absence of therapy
  • the method includes administering a therapeutically effective amount of a composition to a subject who is chemoresistant that includes at least one agent that decreases the biological activity of at least EGFR, FGFR-2 and/or MYC.
  • the method includes administering a therapeutically effective amount of a composition to a subject who is chemoresistant (for example, has acquired or intrinsic chemoresistance) that includes at least one agent that decreases the biological activity of at least one ES marker or MYC target (such as one or more ES markers provided in Table 10 or one or more MYC targets provided in Table 7).
  • a 100% reduction in biological activity is not required; in some examples decreases of at least 20%, at least 30%, at least 50%, at least 75%, at least 80%, or at least 95% relative to no treatment can be sufficient to increase the chemosensitivity of an epithelial cancer).
  • agents are administered to decrease the biological activity of EGFR, FGFR-2 and MYC.
  • Such chemotherapy sensitivity-related molecules include, for instance, nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins.
  • Specific genes include those that encode EGFR, FGFR-2, and MYC as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby) whose expression is upregulated in cancer, such as epithelial cancer including gastric cancer.
  • Additional specific genes include those that encode one or more ES marker or MYC target, as well as fragments of the full-length genes, cDNAs, or mRNAs (and proteins encoded thereby) whose expression is upregulated in cancer, such as epithelial cancer including gastric cancer.
  • the agent can be an inhibitor such as a siRNA or an antibody to one or more of the chemotherapy sensitivity-related molecules, for example, to decrease expression or activity of a gene/protein that is increased in chemoresistance, including EGFR, FGFR-2, MYC, ES markers, and MYC targets.
  • a siRNA siRNA
  • a gene/protein that is increased in chemoresistance, including EGFR, FGFR-2, MYC, ES markers, and MYC targets.
  • Chemoresistance is a complex phenomenon that involves a change in the expression and biological activity of several genes or gene products.
  • the genes or gene families that are expressed differentially in chemoresistant subjects can be used as molecular targets for agents allowing a subject's sensitivity/responsiveness to a chemotherapeutic agent to be increased.
  • inhibiting chemotherapy sensitivity-related molecules that are up-regulated in chemoresistant tumors can be used to treat a tumor.
  • Inhibition of a chemotherapy sensitivity- related molecule does not require 100% inhibition, but can include at least a reduction if not a complete inhibition of cell growth or differentiation associated with a specific pathological condition.
  • Treatment of a tumor by reducing the activity or expression of chemoresistant molecules, including EGFR, FGFR-2, MYC, ES markers, and MYC targets can include delaying the development of the tumor in a subject (such as preventing metastasis of a tumor) by increasing the responsiveness of the tumor to the given chemotherapeutic agent.
  • Treatment of a tumor also includes reducing signs or symptoms associated with the presence of such a tumor (for example by reducing the size or volume of the tumor or a metastasis thereof) by increasing the responsiveness of the tumor to the given chemotherapeutic agent.
  • Such reduced growth can in some examples decrease or slow metastasis of the tumor, or reduce the size or volume of the tumor by at least 10%, at least 20%, at least 50%, or at least 75%.
  • chemotherapy sensitivity-related molecules up-regulated in chemoresistant samples can be inhibited to treat cancer, such as epithelial cancer including gastric cancer, by increasing the responsiveness of the cancer to a chemotherapeutic agent, such as a chemotherapeutic agent including CF.
  • inhibition of chemotherapy sensitivity-related molecules increased with chemoresistance includes reducing the invasive activity of the tumor in the subject, for example by reducing the ability of the tumor to metastasize by increasing the responsiveness of the tumor to a given chemotherapeutic agent.
  • treatment using the methods disclosed herein prolongs the time of survival of the subject.
  • An agent for decreasing biological activity of EGFR, FGFR-2, MYC, ES markers or MYC targets can be one that binds with high affinity to one of the genes or gene products, but does not substantially bind to another gene or gene product.
  • an agent binds to one or more genes that encode EGFR, FGFR- 2, MYC, ES markers or MYC targets which are upregulated, thereby reducing or inhibiting expression of the one or more genes.
  • the agent interferes with gene expression (transcription, processing, translation, post-translational modification), such as, by interfering with the gene's mRNA and blocking translation of the gene product or by post-translational modification of a gene product, or by causing changes in intracellular localization.
  • an agent binds to an EGFR, FGFR-2, MYC, ES marker, or MYC target protein with a binding affinity in the range of 0.1 to 20 nM.
  • the agent is an antagonist (such as a commercially available EGFR, FGFR-2, MYC, ES marker, and/or MYC target antagonist) and is used to inhibit the activity or expression of chemotherapy sensitivity-related molecules that are up-regulated in a chemoresistant tumor, such as a chemoresistant epithelial tumor (e.g., gastric tumor).
  • specific agents include, but are not limited to siRNA, antibodies, ligands, recombinant proteins, peptide mimetics, and soluble receptor fragments.
  • a specific binding agent is a siRNA.
  • Methods of making siRNA that can be used clinically are known in the art.
  • Particular siRNAs and methods that can be used to produce and administer them are described in detail below.
  • siRNA hybridize to molecules that encode EGFR, FGFR-2, MYC, ES markers, and/or MYC targets, with high specificity.
  • a specific binding agent is an antibody, such as a monoclonal or polyclonal antibody, for example a humanized antibody.
  • an antibody such as a monoclonal or polyclonal antibody, for example a humanized antibody.
  • Methods of making antibodies that can be used clinically are known in the art. Particular antibodies and methods that can be used to produce them are described in detail below.
  • Antibodies for EGFR, FGFR-2, MYC, ES markers, and MYC targets can also be obtained from commercially available sources, such as from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).
  • small molecular weight inhibitors or antagonists of EGFR, FGFR-2, MYC, ES marker, and MYC target proteins can be used to regulate chemosensitivity.
  • Agents can be therapeutic, for example by reducing or inhibiting the biological activity of a nucleic acid or protein whose activity is detrimental.
  • an agent that binds with high affinity to a gene that encodes EGFR, FGFR- 2, MYC, an ES marker, or a MYC target may substantially reduce the biological function of the gene or gene product (for example, the ability of the gene or gene product to impart chemoresistance, to a tumor cell).
  • an agent that binds with high affinity to one or more of EGFR, FGFR-2, MYC, ES marker, and MYC target proteins may substantially reduce the biological function of the proteins (for example, the ability of the proteins to promote chemoresistance).
  • Such agents can be administered in therapeutically effective amounts to subjects in need thereof, such as a subject having cancer, such as gastric cancer that is chemoresistant.
  • subjects are initially screened to determine if they are likely to respond to chemotherapy by use of the disclosed gene expression profiles (as discussed in detail above).
  • the disclosed gene expression profiles can be used to determine if a subject with epithelial cancer (such as gastric cancer) is likely to be chemoresistant (for example, intrinsic or acquired chemoresistance) or chemosensitive.
  • a subject that is likely to be chemoresistant or chemosensitive is selected.
  • Subjects that are chemosensitive can receive standard chemotherapy.
  • Subjects that are chemoresistant can receive any of the therapies disclosed herein ⁇ e.g., those that reduce the biological activity of EGFR, FGFR-2, MYC, ES markers, and/or MYC targets).
  • a tumor is an abnormal growth of tissue that results from excessive cell division.
  • a particular example of a tumor is cancer.
  • the current application provides methods for the treatment (such as the prevention or reduction of metastasis) of tumors (such as cancers) by altering a tumor's response to a chemotherapeutic agent.
  • the tumor is treated in vivo, for example in a mammalian subject, such as a human subject.
  • Exemplary tumors that can be treated using the disclosed methods include, but are not limited to epithelial tumors, such as gastric cancer or bladder cancer, including metastases of such tumors to other organs.
  • compositions including one or more therapeutic agents that reduce or inhibit the biological activity of EGFR, FGFR-2, MYC, ES marker, and/or MYC target peptides or nucleic acids encoding such peptides, and further contemplates administering such therapeutics to subjects in need thereof, such as to subjects having chemoresistant gastric tumors.
  • subjects that are identified by methods disclosed herein to have chemoresistant tumors such as chemoresistant epithelial cancers ⁇ e.g.
  • chemoresistant gastric cancers are administered (at therapeutic doses) therapeutic agents that reduce or inhibit the biological activity of EGFR, FGFR-2, MYC and/or one or more ES markers or MYC targets, thereby rendering the tumor more chemosensitive.
  • a therapeutic agent that reduces or inhibits the biological activity of EGFR, FGFR-2, MYC and/or one or more ES markers or MYC targets is administered prior to, concurrently or subsequent to the delivery of the chemotherapeutic agent, such as a CF chemotherapy. Delivery systems and treatment regimens useful for such agents are known and can be used to administer these agents as therapeutics. In addition, representative embodiments are described below. 1. Administration of Nucleic Acid Molecules
  • a therapeutic molecule is a nucleic acid molecule (such as an siRNA, shRNA, antisense oligonucleotide, ribozyme or other inhibitory nucleic acid specific for a gene that is upregulated in chemoresistant gastric cancer)
  • administration of the nucleic acid may be achieved in a variety of ways. All forms of nucleic acid delivery are contemplated by this disclosure, including, without limitation, synthetic oligos, naked DNA, naked RNA (such as capped RNA), and plasmid or viral vectors (which may or may not be integrated into a target cell genome).
  • an expressible nucleic acid can be administered by use of a viral vector (see U.S. Patent No.
  • the expressible nucleic acid can be introduced into a host cell (such as a stem cell, e.g., a stem cell capable of neural differentiation) for expression of a polypeptide therapeutic in the host cell.
  • a host cell such as a stem cell, e.g., a stem cell capable of neural differentiation
  • transfected/transformed host cells can be transplanted into a subject.
  • a nucleic acid molecule can be incorporated within host cell DNA, for example, by homologous or non-homologous recombination, for stably expressing a therapeutic.
  • Expression vectors are commonly available that provide, for instance, constitutive, regulated, or cell/tissue-specific expression of a transcribable nucleic acid (e.g., a nucleic acid encoding a chemotherapy sensitivity-related molecule polypeptide) included in the expression vector. All these vectors achieve the basic goal of delivering into the target cell a heterologous nucleic acid sequence and control elements needed for transcription.
  • the vector pcDNA which includes a strong viral promoter (CMV), is an example of an expression vector for constitutive expression of a heterologous DNA.
  • Certain retroviral vectors (such as pRETRO- ON, Clontech) also use the constitutive CMV promoter but have the advantages of entering cells without any transfection aid, integrating into the genome of target cells only when the target cell is dividing.
  • Regulated expression vectors include control elements that permit expression of an operably linked nucleic acid only when a corresponding regulator molecule (such as tetracycline or steroid hormones) is present.
  • Exemplary regulated vectors include pMAM-neo (Clontech) or pMSG (Pharmacia), which use the steroid-regulated MMTV-LTR promoter, or pBPV (Pharmacia), which includes a metallothionein-responsive promoter.
  • Numerous cell/tissue-specific expression vectors are also available for expression of heterologous nucleic acids in any of a variety of tissues or cell types.
  • Viral vectors which are derived from various viral genomes, are similarly numerous and commercially available.
  • Exemplary viral vectors are derived from retroviruses (such as lentivirus), adenovirus, herpes simplex virus (HSV; Margolskee et al, MoI Cell. Biol. 8:2837-2847, 1988), adeno-associated virus (McLaughlin et al, J. Virol. 62:1963-1973, 1988), polio virus and vaccinia virus (Moss et al., Annu. Rev. Immunol. 5:305-324, 1987).
  • retroviruses such as lentivirus
  • HSV herpes simplex virus
  • HSV herpes simplex virus
  • McLaughlin et al J. Virol. 62:1963-1973, 1988
  • polio virus and vaccinia virus Moss et al., Annu. Rev. Immunol. 5:305-324, 1987.
  • retroviral vectors are derived from lentiviruses, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • Multiple teachings concerning viral vectors are available, e.g., Anderson, Science, 226:401-409, 1984; Hughes, Curr. Comm. MoI. Biol., 71:1-12, 1988; Friedman, Science, 244:1275-1281, 1989 and Anderson, Science, 256:608-613, 1992.
  • Some viral vectors are replication-deficient and/or non-infective.
  • Non-limiting representative neurotrophic viral vectors include herpes simplex viral vectors (see, e.g., U.S. Pat. No. 5,673,344) and adenoviral vectors (see, e.g., Barkats et al, Prog. Neurobiol, 55:333-341, 1998), or AAV or lentiviral vectors pseudotyped with rabies-G glycoprotein (Mazarakis et al, Human MoI. Genet , 10:2109-2121, 2001; Azzouz, et al., J. Neurosci., 22:10302-10312, 2002; Azzouz, et al., Nature, 429:413-417, 2004).
  • lipidic and liposome-mediated gene delivery has recently been used successfully for transfection with various genes (for reviews, see Templeton and Lasic, MoI. Biotechnol, 11:175 180, 1999; Lee and Huang, Crit. Rev. Ther. Drug Carrier Syst , 14:173-206, 1997; and Cooper, Semin. Oncol , 23:172-187, 1996).
  • cationic liposomes have been analyzed for their ability to transfect monocytic leukemia cells, and shown to be a viable alternative to using viral vectors (de Lima et al, MoI. Membr. Biol., 16:103-109, 1999).
  • Such cationic liposomes can also be targeted to specific cells through the inclusion of, for instance, monoclonal antibodies or other appropriate targeting ligands (Kao et al. , Cancer Gene Ther. , 3:250-256, 1996).
  • therapeutic agents comprising peptides may be delivered by administering to the subject a nucleic acid encoding the peptide, in which case the methods discussed in the section entitled "Administration of Nucleic Acid Molecules" should be consulted.
  • peptide therapeutic agents may be isolated from various sources and administered directly to the subject. For example, a peptide may be isolated from a naturally occurring source. Alternatively, a nucleic acid encoding the peptide may be expressed in vitro, such as in an E. coli expression system, as is well known in the art, and isolated in amounts useful for therapeutic compositions.
  • commercially available inhibitors of EGFR, FGFR-2, MYC, ES markers, and/or MYC targets are administered to a subject to increase the sensitivity of the tumor prior to, concurrent with or subsequent to the administration of chemotherapy, such as CF chemotherapy.
  • chemotherapy such as CF chemotherapy.
  • FGFR-2, MYC and one or more ES markers or MYC targets can be administered to a subject with a chemoresistant tumor, such as a chemoresistant epithelial tumor (e.g. , gastric tumor).
  • a chemoresistant tumor such as a chemoresistant epithelial tumor (e.g. , gastric tumor).
  • EGFR inhibitor therapies include at least two drug classes: EGFR antibody therapies (such as, cetuximab (ErbituxTM), panitumumab (VectibixTM), IMC- 11F8 (Imclone), matuzumab (Merck_KGA)) and tyrosine kinase inhibitors ("TKIs") (such as gefitinib (IressaTM), erlotinib (TarcevaTM), lapatinib ditosylate (GlaxoSmithKline), HKI-272 (Wyeth), AEE788 (Novartis), vandetanib (ZactimaTM; Astrazeneca), XL647 (Exelixis), BMS-599626 (Bristol-Myers Squibb), BIBW 2992 (Boehringer Ingelheim)).
  • EGFR antibody therapies such as, cetuximab (ErbituxTM), panitumumab (VectibixTM
  • EGFR antibody therapies typically are directed to the EGFR external domain and block binding of an EGFR ligand (such as EGF) to the receptor; thereby, inhibiting EGFR activation.
  • TKIs work by inhibiting the intracellular kinase domain of EGFR, which also inhibits EGFR activation.
  • Some method embodiments involve one or both of the foregoing classes of EGFR inhibitors. Particular method embodiments involve administering at least one of these inhibitors either alone or in combination with FGFR-2 and MYC inhibitors. In one example, FGFR-2 inhibitors such as PD173074 are administered either alone or in combination with EGFR inhibitors or MYC inhibitors.
  • one or more ES marker inhibitors or MYC target inhibitors is administered, either alone or in combination with EGFR, FGFR2, and/or MYC inhibitors.
  • therapeutic agents are administered prior to, concurrently or subsequent to administration of one or more chemotherapeutic agents.
  • a subject is administered a therapeutic agent prior to, concurrently or subsequent to administration of a combined CF chemotherapy.
  • concentration of cisplatin and fluorouracil can be determined by one of ordinary skill in the art.
  • the concentration of cisplatin administered by IV ranges from 10 mg/m 2 to 100 mg/m 2 , such as at least 20 mg/m 2 , at least 30 mg/m , at least 40 mg/m , at least 50 mg/m , at least 60 mg/m , at least 70 mg/m 2 , at least 80 mg/m 2 , at least 90 mg/m 2 .
  • the concentration of fluorouracil administered by IV ranges from 100 mg/m 2 to 2000 mg/m 2 , such as at least 200 mg/m 2 , at least 300 mg/m 2 , at least 400 mg/m 2 , at least 500 mg/m 2 , at least 600 mg/m 2 , at least 700 mg/m 2 , at least 800 mg/m 2 , at least 900 mg/m 2 , at least 1000 mg/m 2 , at least 1100 mg/m 2 , at least 1200 mg/m 2 , at least 1400 mg/m 2 , at least 1600 m mg/m 2 , at least 1800 mg/m 2 .
  • a subject receives 60 mg/m 2 IV on day one and fluorouracil 1,000 mg/m 2 , IV on days 1-5 of a 3-week schedule.
  • the duration of the treatment can be altered as needed, such as the number of days of treatment can be shortened or lengthened.
  • Subjects can continue the therapy indefinitely until unacceptable toxicities are experienced.
  • Additional chemotherapy regimens for gastric cancers include DCF (docetaxel, cisplatin, and 5-fluorouracil) and ECF (epirubicin, cisplatin, and 5- fluorouracil).
  • DCF docetaxel, cisplatin, and 5-fluorouracil
  • ECF epirubicin, cisplatin, and 5- fluorouracil
  • the disclosed CF regimens can be used for a variety of epithelial cancers originating from uterine cervix, liver, pancreas, esophagus, head and neck. It is one of reference regimens prescribed for these cancers at their metastatic stage, and, concurrently with radiation, at locally-advanced stage.
  • DNA intercalators and cross-linking agents can be administered to a subject in addition to or instead of cisplatin including, without limitation, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof.
  • cisplatin including, without limitation, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof.
  • DNA synthesis inhibitors can be administered to a subject in addition to or instead of fluorouracil, including, but not limited to, methotrexate, 5-fluoro-5'-deoxyuridine and analogs thereof.
  • Methods of administering a disclosed therapeutic include, but are not limited to, intrathecal, intradermal, intramuscular, intraperitoneal (ip), intravenous (iv), subcutaneous, intranasal, epidural, intradural, intracranial, intraventricular, and oral routes.
  • a therapeutic may be administered by any convenient route, including, for example, infusion or bolus injection, topical, absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, vaginal mucosa and the like), ophthalmic, nasal, and transdermal, and may be administered together with other biologically active agents. Administration can be systemic or local.
  • injection may be facilitated by a catheter, for example, attached to a reservoir.
  • a pump may be used (see, e.g., Langer Science 249, 1527, 1990; Sefton Crit. Rev. Biomed. Eng.
  • administration is achieved by intravenous, intradural, intracranial, intrathecal, or epidural infusion of a therapeutic using a transplanted minipump.
  • minipump may be transplanted in any location that permits effective delivery of the therapeutic agent to the target site; for instance, a minipump may be transplanted near the tumor.
  • administration can be by direct injection at the site (or former site) of a tissue that is to be treated, such as the gastric tumor site.
  • a therapeutic is delivered in a vesicle, in particular liposomes (see, e.g., Langer, Science 249, 1527, 1990; Treat et al , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N. Y., pp. 353-365, 1989).
  • a therapeutic agent can be delivered in a controlled release system.
  • polymeric materials can be used (see, e.g., Ranger et al, Macromol. Sci. Rev. Macromol. Chem.
  • the vehicle in which an agent is delivered can include pharmaceutically acceptable compositions known to those with skill in the art.
  • therapeutic agents disclosed herein are contained in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government or listed in the U.S.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions, blood plasma medium, aqueous dextrose, and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the medium may also contain conventional pharmaceutical adjunct materials such as for example, pharmaceutically acceptable salts to adjust the osmotic pressure, lipid carriers such as cyclodextrins, proteins such as serum albumin, hydrophilic agents such as methyl cellulose, detergents, buffers, preservatives and the like.
  • pharmaceutically acceptable salts to adjust the osmotic pressure
  • lipid carriers such as cyclodextrins, proteins such as serum albumin
  • hydrophilic agents such as methyl cellulose
  • detergents such as buffers, preservatives and the like.
  • Examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.
  • the therapeutic if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the therapeutic can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • the therapeutic can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • parenteral pharmaceutical carriers can be found in Remington: The Science and Practice of Pharmacy (19th Edition, 1995) in chapter 95.
  • Embodiments of other pharmaceutical compositions are prepared with conventional pharmaceutically acceptable counterions, as would be known to those of skill in the art.
  • Therapeutic preparations will contain a therapeutically effective amount of at least one active ingredient, preferably in purified form, together with a suitable amount of carrier so as to provide proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and biologically active or inactive compounds (or both), such as antineoplastic agents and conventional nontoxic pharmaceutically acceptable carriers, respectively.
  • the ingredients in various embodiments are supplied either separately or mixed together in unit dosage form, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, or suspensions, or as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to administration.
  • the amount of the therapeutic that will be effective depends on the nature of the disorder or condition to be treated, as well as the stage of the disorder or condition. Effective amounts can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and should be decided according to the judgment of the health care practitioner and each patient's circumstances. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.
  • the therapeutic agents of the present disclosure can be administered at about the same dose throughout a treatment period, in an escalating dose regimen, or in a loading-dose regime (for example, in which the loading dose is about two to five times the maintenance dose).
  • the dose is varied during the course of a treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to the therapy, and/or other factors as judged by one of ordinary skill in the art.
  • long-term treatment with a disclosed therapeutic is contemplated, for instance in order to have sustained decreased expression or activity of a chemotherapy sensitivity-related molecule (e.g.
  • the method includes daily administration of at least 1 ⁇ g of the composition to the subject (such as a human subject).
  • a human can be administered at least 1 ⁇ g or at least 1 mg of the therapeutic composition (e.g., RNAi or antibody) daily, such as 10 ⁇ g to 100 ⁇ g daily, 100 ⁇ g to 1000 ⁇ g daily, for example 10 ⁇ g daily, 100 ⁇ g daily, or 1000 ⁇ g daily.
  • the subject is administered at least 1 ⁇ g (such as 1-100 ⁇ g) intravenously of the composition including a binding agent that specifically binds to one or more of the disclosed chemotherapy sensitivity-related molecules including EGFR, FGFR-2, MYC, ES markers, or MYC target proteins or molecules that encode EGFR, FGFR-2, MYC, ES markers, or MYC target proteins.
  • the subject is administered at least 1 mg intramuscularly (for example in an extremity) of such composition.
  • the dosage can be administered in divided doses (such as 2, 3, or 4 divided doses per day), or in a single dosage daily.
  • the subject is administered the therapeutic composition that includes an agent for one or more of the disclosed chemotherapy sensitivity-related molecules on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years.
  • the subject is administered the therapeutic composition that a binding agent specific for one or more of the disclosed chemotherapy sensitivity-related molecules daily for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.
  • the subject prior to, during, or following administration of a therapeutic amount of an agent that reduces or inhibits chemoresistance by decreasing the biological activity of EGFR, FGFR-2, MYC, ES markers, and/or MYC targets the subject can receive one or more other therapies.
  • the subject receives one or more treatments to remove or reduce the tumor prior to administration of a therapeutic amount of a composition including an agent specific for one or more of the disclosed chemotherapy sensitivity-related molecules, such as commercially available inhibitors of EGFR, FGFR-2, MYC, ES markers, or MYC targets.
  • Such therapies include, but are not limited to, surgical treatment for removal or reduction of the tumor (such as surgical resection, cryotherapy, or chemoembolization), as well as anti-tumor pharmaceutical treatments which can include radiotherapeutic agents, anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents.
  • additional therapeutic agents include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination. Methods and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.
  • Microtubule binding agent refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division.
  • microtubule binding agents that can be used in conjunction with the disclosed therapy include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin. Analogs and derivatives of such compounds also can be used and are known to those of ordinary skill in the art. For example, suitable epothilones and epothilone analogs are described in International Publication No.
  • Taxoids such as paclitaxel and docetaxel, as well as the analogs of paclitaxel taught by U.S. Patent Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.
  • Suitable DNA and/or RNA transcription regulators including, without limitation, actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof also are suitable for use in combination with the disclosed therapies.
  • Suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof.
  • Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5- azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.
  • Kinase inhibitors include Gleevac, Iressa, and Tarceva that prevent phosphorylation and activation of growth factors.
  • anti-tumor agents for example anti-tumor agents, that may or may not fall under one or more of the classifications above, also are suitable for administration in combination with the disclosed therapies.
  • agents include adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.
  • the therapeutic composition (such as one including one or more agents that decrease the biological activity of EGFR, FGFR-2, MYC, ES marker, or MYC target) is injected into the subject in the presence of an adjuvant.
  • An adjuvant is an agent that when used in combination with an immunogenic agent augments or otherwise alters or modifies a resultant immune response.
  • an adjuvant increases the titer of antibodies induced in a subject by the immunogenic agent.
  • the one or more peptides are administered to the subject as an emulsion with a IFA and sterile water for injection (for example an intravenous or intramuscular injection).
  • Incomplete Freund's Adjuvant can be used as the Freund's Incomplete Adjuvant (IFA) (Fairfield, NJ).
  • IFA is provided in 3 ml of a mineral oil solution based on mannide oleate (Montanide ISA-51).
  • the peptide(s) is mixed with the Montanide ISA.51 and then administered to the subject.
  • Other adjuvants can be used, for example, Freund's complete adjuvant, B30-MDP, LA-15-PH, montanide, saponin, aluminum hydroxide, alum, lipids, keyhole lympet protein, hemocyanin, a mycobacterial antigen, and combinations thereof.
  • the subject receiving a therapeutic composition is also administered interleukin-2 (IL-2), for example via intravenous administration.
  • IL-2 interleukin-2
  • IL-2 Chiron Corp., Emeryville, CA
  • IL-2 is administered at a dose of at least 500,000 IU/kg as an intravenous bolus over a 15 minute period every eight hours beginning on the day after administration of the peptides and continuing for up to 5 days. Doses can be skipped depending on subject tolerance.
  • compositions can be co-administered with a fully human antibody to cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4).
  • anti-CTLA-4 cytotoxic T-lymphocyte antigen-4
  • subjects receive at least 1 mg/kg anti-CTLA-4 (such as 3 mg/kg every 3 weeks or 3 mg/kg as the initial dose with subsequent doses reduced to 1 mg/kg every 3 weeks).
  • At least a portion of the tumor (such as a metastatic tumor) is surgically removed (for example via cryotherapy), irradiated, chemically treated (for example via chemoembolization) or combinations thereof, prior to administration of the disclosed therapies (such as administration of a composition for one or more of the disclosed chemotherapy sensitivity-related molecules).
  • a subject having a metastatic tumor can have all or part of the tumor surgically excised prior to administration of the disclosed therapies (such as one including a binding agent specific for one or more of the disclosed chemotherapy sensitivity-related molecules).
  • one or more chemotherapeutic agents is administered following treatment with a binding agent specific for one or more of the disclosed chemotherapy sensitivity -related molecules.
  • the subject has a metastatic tumor and is administered radiation therapy, chemoembolization therapy, or both concurrently with the administration of the disclosed therapies (such as one including agents specific for one or more of the disclosed chemotherapy sensitivity-related molecules administered prior to, concurrently with or following administration of chemotherapeutic agents, such as CF therapy).
  • the disclosed therapies such as one including agents specific for one or more of the disclosed chemotherapy sensitivity-related molecules administered prior to, concurrently with or following administration of chemotherapeutic agents, such as CF therapy.
  • siRNAs are species of siRNAs.
  • One of ordinary skill in the art can readily generate siRNAs which specifically bind to one or more of the disclosed chemotherapy sensitivity-related molecules that are upregulated in chemorefractory or chemoresistant gastric cancers, including siRNAs that specifically bind to molecules that encode EGFR, FGFR-2, MYC, ES markers, or MYC targets.
  • commercially available kits such as siRNA molecule synthesizing kits from PROMEGA ® (Madison, WI) or AMBION ® (Austin, TX) may be used to synthesize siRNA molecules.
  • siRNAs are obtained from commercial sources, such as from QIAGEN ® Inc (Germantown, MD), INVITROGEN ® (Carlsbad, CA), AMBION (Austin, TX), DHARMACON ® (Lafayette, CO) or OPENBIOSYSTEMS ® (Huntsville, AL).
  • expression vectors are employed to express the at least one siRNA molecule.
  • an expression vector can include a nucleic acid sequence encoding at least one siRNA molecule corresponding to at least one molecule that encodes EGFR, FGFR-2, MYC, ES markers, or MYC targets that are upregulated in chemoresistant gastric cancers.
  • the vector contains a sequence(s) encoding both strands of a siRNA molecule comprising a duplex.
  • the vector also contains sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a siRNA molecule.
  • Non- limiting examples of such expression vectors are described in Paul et al.
  • siRNA molecules include a delivery vehicle, including inter alia liposomes, for administration to a subject, carriers and diluents and their salts, and can be present in pharmaceutical compositions.
  • Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other delivery vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (see, for example, O'Hare and Normand, International PCT Publication No. WO 00/53722).
  • the nucleic acid/vehicle combination can be locally delivered by direct injection or by use of an infusion pump.
  • Direct injection of the nucleic acid molecules of the disclosure can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described by Barry et al, International PCT Publication No. WO 99/31262.
  • Other delivery routes but are not limited to, oral delivery (such as in tablet or pill form), intrathecal or intraperitoneal delivery.
  • intraperitoneal delivery can take place by injecting the treatment into the peritoneal cavity of the subject in order to directly deliver the molecules to the tumor site.
  • nucleic acid delivery and administration are provided in Sullivan et al, PCT WO 94/02595, Draper et al, PCT WO93/23569, Beigelman et al, PCT WO99/05094, and Klimuk et al, PCT WO99/04819, all of which are incorporated by reference herein.
  • certain siRNA molecules can be expressed within cells from eukaryotic promoters. Those skilled in the art will recognize that any nucleic acid can be expressed in eukaryotic cells using the appropriate DNA/RNA vector.
  • siRNA molecules can be expressed from transcription units (see for example, Couture et al, 1996, TIG 12:510) inserted into DNA or RNA vectors.
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • siRNA expressing viral vectors can be constructed based on, for example, but not limited to, adeno-associated virus, retrovirus, adenovirus, lentivirus or alphavirus.
  • pol III based constructs are used to express nucleic acid molecules of the invention (see for example, Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886).
  • the recombinant vectors capable of expressing the siRNA molecules can be delivered as described above, and persist in target cells.
  • viral vectors can be used that provide for transient expression of nucleic acid molecules.
  • Such vectors can be repeatedly administered as necessary.
  • the siRNA molecule interacts with the target mRNA and generates an RNAi response.
  • Delivery of siRNA molecule expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.
  • antibodies which specifically bind to the disclosed chemotherapy sensitivity-related molecules including EGFR, FGFR-2, MYC, ES markers, and MYC targets. These antibodies can be monoclonal or polyclonal. They can be chimeric or humanized. Any functional fragment or derivative of an antibody can be used including Fab, Fab', Fab2, Fab'2, and single chain variable regions. So long as the fragment or derivative retains specificity of binding for the chemotherapy sensitivity-related molecule it can be used in the methods provided herein. Antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions.
  • monoclonal antibodies are generated to the chemotherapy sensitivity-related molecules including EGFR, FGFR-2, MYC, or one or more ES markers or MYC targets. These monoclonal antibodies each include a variable heavy (V H ) and a variable light (V L ) chain and specifically bind to the specific chemotherapy sensitivity -related molecules.
  • the antibody can bind the specific chemotherapy sensitivity-related molecules with an affinity constant of at least 10 6 M “1 , such as at least 10 7 M “1 , at least 10 8 M “1 , at least 5 x 10 8 M “1 , or at least 10 9 M- 1 .
  • the specific antibodies can include a V L polypeptide having amino acid sequences of the complementarity determining regions (CDRs) that are at least about 90% identical, such as at least about 95%, at least about 98%, or at least about 99% identical to the amino acid sequences of the specific chemotherapy sensitivity- related molecules and a V H polypeptide having amino acid sequences of the CDRs that are at least about 90% identical, such as at least about 95%, at least about 98%, or at least about 99% identical to the amino acid sequences of the specific chemotherapy sensitivity -related molecules.
  • CDRs complementarity determining regions
  • the sequence of the specificity determining regions of each CDR is determined. Residues that are outside the CDR (non-ligand contacting sites) are substituted. For example, in any of the CDR sequences, at most one, two or three amino acids can be substituted.
  • the production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is well known in the art.
  • humanized antibodies can be routinely produced.
  • the antibody or antibody fragment can be a humanized immunoglobulin having CDRs from a donor monoclonal antibody that binds one of the disclosed chemotherapy sensitivity-related molecules and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks.
  • the humanized immunoglobulin specifically binds to one of the disclosed chemotherapy sensitivity- related molecules with an affinity constant of at least 10 7 M "1 , such as at least 10 8 M “1 at least 5 x 10 8 M “1 or at least 10 9 M "1 .
  • human monoclonal antibodies to the disclosed chemotherapy sensitivity-related including EGFR, FGFR-2, MYC or one or more ES markers or MYC targets are produced.
  • Human monoclonal antibodies can be produced by transferring donor complementarity determining regions (CDRs) from heavy and light variable chains of the donor mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity.
  • CDRs donor complementarity determining regions
  • antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody.
  • mouse monoclonal antibodies when used therapeutically, the development of human anti-mouse antibodies (HAMA) leads to clearance of the murine monoclonal antibodies and other possible adverse events.
  • HAMA human anti-mouse antibodies
  • Chimeric monoclonal antibodies, with human constant regions, humanized monoclonal antibodies, retaining only murine CDRs, and "fully human" monoclonal antibodies made from phage libraries or transgenic mice have all been used to reduce or eliminate the murine content of therapeutic monoclonal antibodies.
  • the antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgGi, IgG 2 , IgG 3 and IgG 4 .
  • the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
  • the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
  • Human framework regions, and mutations that can be made in a humanized antibody framework regions, are known in the art (see, for example, U.S. Patent No. 5,585,089).
  • Antibodies such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which include a heavy chain and light chain variable region and are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with their antigen or receptor.
  • fragments include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab') 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by
  • Fv antibodies are typically about 25 kDa and contain a complete antigen- binding site with three CDRs per each heavy chain and each light chain.
  • the V H and the V L can be expressed from two individual nucleic acid constructs in a host cell. If the V H and the V L are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker.
  • the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
  • the Fv fragments include V H and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing scFvs are known in the art (see Whitlow et al, Methods: a Companion to Methods in
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the V H and the V L regions, and will retain the charge characteristics of the residues in order to preserve the low pi and low toxicity of the molecules.
  • Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the V H and the V L regions to increase yield.
  • Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art.
  • the following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • kits that can be used to diagnose, prognose, or treat a gastric or other type of epithelial cancer that has increased expression of chemotherapy-sensitivity related molecules, including EGFR, FGFR-2, MYC, ES markers, and MYC target proteins or nucleic acids that encode EGFR, FGFR-2, MYC, ES markers and MYC target proteins.
  • the disclosed kits can include instructional materials disclosing means of use of the compositions in the kit.
  • the instructional materials can be written, in an electronic form (such as a computer diskette or compact disk) or can be visual (such as video files). Instructions may also provide calibration curves or charts to compare with the determined (such as experimentally measured) values.
  • kits are provided that can be used in the therapies and diagnostic assays disclosed herein.
  • kits can include at least one or more of the disclosed therapeutic compositions (such as a composition including one or more of the siRNAs directed to at least EGFR, FGFR-2, and MYC and in some examples also one or more ES markers or MYC target molecules upregulated in chemoresistant gastric cancer), one or more of the disclosed gene profile signatures, or combinations thereof.
  • the kits can include other agents to facilitate the particular application for which the kit is designed.
  • kits for treating a gastric cancer that is chemoresistant.
  • such kits can include one or more of the disclosed therapeutic compositions (such as a composition including a siRNA or antibody specific for one or more of the chemotherapy sensitivity-related molecules including EGFR, FGFR-2, MYC, ES markers, and MYC target proteins or nucleic acids that encode EGFR, FGFR-2, MYC, ES markers, and MYC target proteins that are upregulated in chemoresistant epithelial cancers, such as gastric cancer).
  • the disclosed therapeutic compositions such as a composition including a siRNA or antibody specific for one or more of the chemotherapy sensitivity-related molecules including EGFR, FGFR-2, MYC, ES markers, and MYC target proteins or nucleic acids that encode EGFR, FGFR-2, MYC, ES markers, and MYC target proteins that are upregulated in chemoresistant epithelial cancers, such as gastric cancer).
  • a kit for detecting one or more of the disclosed chemosensitivity-related molecules including EGFR, FGFR-2 and MYC proteins or nucleic acids that encode EGFR, FGFR-2 and MYC proteins in a biological sample, such as a tissue biopsy, cells isolated from a tissue biopsy, serum, or urine.
  • a kit is provided for detecting one or more of the disclosed chemosensitivity-related molecules including one or more ES markers in a biological sample, such as a tissue biopsy, cells isolated from a tissue biopsy, serum, or urine.
  • kits for detecting one or more of the disclosed chemosensitivity-related molecules including one or more MYC target proteins or nucleic acids that encode one or more MYC target proteins in a biological sample such as tissue biopsy, cells isolated from a tissue biopsy, serum, or urine.
  • Kits for detecting chemosensitivity-related molecules can include one or more probes that specifically bind to the molecules, such as probes that specifically bind to EGFR, FGFR-2, MYC, ES markers, or MYC target proteins or nucleic acids that encode EGFR, FGFR-2, MYC, ES markers, or MYC target proteins.
  • a kit includes an array with at least EGFR, FGFR-2 and MYC proteins or nucleic acids that encode EGFR, FGFR-2 and MYC proteins and controls, such as positive and negative controls.
  • a kit includes an array with at least one or more ES marker proteins or nucleic acid that encodes one or more ES marker proteins and controls, such as positive and negative controls.
  • a kit includes an array with at least one or more MYC target protein or nucleic acid that encodes one or more MYC target protein and controls, such as positive and negative controls.
  • kits include primers that specifically bind to nucleic acids that encode EGFR, FGFR-2 and MYC proteins and/or primers that specifically bind to nucleic acids that encode one or more ES marker proteins or MYC target proteins. Such kits can further include reagents required to perform PCR or quantitative PCR. In other examples, kits include antibodies that specifically bind to EGFR,
  • FGFR-2, MYC, ES markers and/or MYC target proteins are labeled (for example, with a fluorescent, radioactive, or an enzymatic label).
  • a diagnostic kit can additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like), as well as buffers and other reagents routinely used for the practice of a particular diagnostic method.
  • Tissue samples were collected by endoscopy. Eligibility criteria for the inclusion into the study included the following parameters: 1) age greater than or equal to 18 years; 2) histologically-confirmed gastric carcinoma; 3) clinically-documented distant metastasis (subjects with locally-advanced disease were excluded); 4) no previous or concomitant malignancies other than gastric cancer; 5) no prior history of chemotherapy, either adjuvant or palliative; and 6) adequate function of all major organs. Subjects who were lost to follow-up before completing 6 cycles of chemotherapy, except for documented progressive disease, were also ineligible for this study. Of the subjects contacted, 178 met the eligibility criteria.
  • DNA microarray data of the pretreatment endoscopic biopsy samples from the remaining 96 subjects (53.9%) were used for the expression profiling training set. All subjects in the training cohort were treated by one medical oncologist. Thirty-eight eligible consecutively accrued subjects treated with the CF regimen by a second group of medical oncologists were used for the array validation cohort. Adequate quantity and quality of total RNA were retrieved from 22 (57.9%) of the 38 subject samples collected.
  • cisplatin plus oral capecitabine a fluorouracil pro-drug considered equivalent to fluorouracil
  • Cisplatin plus capecitabine has been shown to be therapeutically equivalent to the CF regimen for metastatic gastric cancer. Tissue procurement and processing were the same for the training and validation samples. Clinico-pathological characteristics of the subject groups are listed in Table 1.
  • Treatment and follow-up Subjects received cisplatin 60 mg/m 2 IV on day 1 and fluorouracil 1,000 mg/m IV on days 1-5 of a 3-week schedule. Subjects continued therapy indefinitely until they experienced unacceptable toxicities or progressive disease was documented.
  • the treatment schedule for fluorouracil could be shortened at the discretion of the oncologist to 3 days instead of 5 days for elderly subjects (greater than or equal to 70 years) or subjects with poor performance status (Eastern Cooperative Oncology Group (ECOG) performance status greater than or equal to T).
  • Chemotherapy doses were reduced, according to toxicities observed in the previous treatment cycles and based upon the subject's nutritional status and performance status. Specific dose modification schemes for the subsequent cycle were left to the discretion of the treating oncologist.
  • Tissue samples were collected and processed as previously described (Kim et al. Biochem Biophys Res Comniun 316(3):781 -789, 2004). Briefly, 10 pieces of biopsy tissue samples were collected during the diagnostic endoscopy procedure, and immediately frozen in liquid nitrogen. Serial cryosections of the biopsy samples embedded in OCT were made, until a representative section containing the majority of the embedded tissue was identified. The corresponding tumor-rich area was manually dissected out of the remaining OCT block in a cryostat, with care taken to avoid contamination of non-neoplastic cells in the tumor samples. RNA was only extracted from samples that contained at least 50% tumor cells, using TRI Reagent (Invitrogen, Carlsbad, CA).
  • Microarray data were normalized by Robust Multichip Average, and analyzed with survival analysis algorithms of BRB Array Tools (version 3.6, National Cancer Institute, available on the World Wide Web at web address linus.nci.nih.gov/BRB-Array Tools.html).
  • the survival analysis tool identifies genes whose expression is correlated with survival time by fitting a proportional hazards model relating survival to the expression of each gene. P values are calculated for each gene to test the hypothesis that survival time is independent of the expression level for that gene.
  • the survival risk groups were constructed using the supervised principal component method of B air and Tibshirani (Bair and Tibshirani PLoS Biol.
  • a prognostic index was computed for each subject by this supervised principal component method, where a high value of the prognostic index corresponds to a high risk of death. If the prognostic index of a sample in the validation set corresponded to the median prognostic index of the training set, the sample was assigned a 50% prognostic index.
  • the number of risk groups and the prognostic index percentile for defining the groups are specified as 2 and 67%, respectively, based on the 67.1% rate of clinical benefit (partial response and stable disease) and 32.9% rate of progressive disease in the training set.
  • a possible reason for the relatively high progressive disease rate in the training group which is higher than the 12-26% progressive disease rates previously reported in phase II/III trials (Van Cutsem et al. J. Clin. Oncol. 24(31): 4991-4997, 2006), may be partly related to more inclusive entry criteria in the present study. Functional categorization of the genes whose expressions were found to be significantly associated with survival was performed using Ingenuity Pathway Analysis (IPA, Ingenuity Systems).
  • This example describes methods used to identify three chemotherapy sensitivity-related molecules that can be used to predict a subject's response to chemotherapy, such as the chemoresponsiveness of a metastatic gastric cancer response to CF chemotherapy.
  • DNA replication/recombination/repair and cancer genes were highly represented categories identified in the poor prognosis signature, according to the IPA algorithm. These data indicate that the up-regulation of DNA repair pathways and oncogene overexpression are highly consistent features of tumors with clinical resistance to CF combination chemotherapy. The functional category cell death was the most represented category in the good-prognosis gene signature.
  • the 3-gene prognostic index performed better than the individual genes alone in calculating hazard ratios (HR), with the HR for the 3-gene signature of 2.65, and HRs of 1.35, 1.65, and 1.44, for, MYC, EGFR, and FGFR2, respectively.
  • Multivariate Cox regression analyses demonstrated that expression of the 3-genes in tumors is a significant predictor for poor survival in the validation cohort with an adjusted HR of 3.15 (95% confidence interval, 1.18-8.38), independent of age (greater than or equal to 70 years vs less than 70 years), performance status (ECOG PS 2-3 vs 0-1), and histological type (diffuse vs intestinal; Table T).
  • the multivariate Cox regression analysis demonstrated that the predicted prognostic index percentile is a significant predictor for poor survival, independent of age, performance status, and histological type (Table T).
  • MYC, EGFR, and FGFR-2 are functionally related to many of the 965 genes within the global poor-prognosis signature, which is particularly enriched for gene involved in DNA repair pathways. For instance, following cisplatin-induced DNA damage, EGFR is imported into the nucleus with Ku70/XRCC5, resulting in increased DNA-dependent protein kinase (DNA-PK) activity involved in DNA repair.
  • DNA-PK DNA-dependent protein kinase
  • XRCC5 and PRKDC encoding a DNA-PK catalytic subunit
  • PRKDC encoding a DNA-PK catalytic subunit
  • various other categories of DNA repair enzymes are up-regulated either directly by MYC or through pathways downstream of EGFR and FGFR2 involving PI3K and ERK (Menssen and Hermeking Proc Natl Acad Sci U.S.A. 99(9): 6274-6279, 2002; and Andrieux et al. Cancer Research 67: 2114-2123, 2007).
  • the disclosed predictive signature can be tested in clinical trials in which metastatic gastric cancer patients, who are predicted to be at high risk using the 3-gene signature, will be treated with a regimen other than CF.
  • This signature can also predict the response of locally-advanced gastric cancers to neoadjuvant chemotherapy, which could be readily tested given that fluorouracil and platinum constitute a backbone for most chemotherapy regimens used for metastatic and operable gastric cancers.
  • the small number of genes in the disclosed signature increases the utility of this predictor in the conduct of clinical trials.
  • the 3- gene signature provides new opportunities to improve the management of gastric cancer subjects as well as subjects with other forms of cancer.
  • a subject's responsiveness to chemotherapy can be predicted prior to providing chemotherapy by detecting increased expression of at least EGFR, FGFR-2 and MYC in a sample obtained from the subject with an epithelial cancer, such as metastatic gastric cancer.
  • the presence of increased expression of EGFR, FGFR-2 and MYC indicates that the cancer has a decreased sensitivity to chemotherapy treatment.
  • the expression product can be RNA or protein.
  • biopsy tissue samples can be collected during a diagnostic endoscopy procedure, and immediately frozen in liquid nitrogen.
  • An RNA expression product can be detected by PCR according to methods known to those of skill in the art.
  • a tissue sample is obtained from a subject identified to have gastric cancer.
  • At least one microgram of RNA is then isolated from the samples that contain at least 50% tumor cells by using TRI Reagent (Invitrogen, Carlsbad, CA).
  • cDNA can then be synthesized from the isolated RNA.
  • PCR is performed in the presence of PCR reagents, cDNA synthesized from the isolated RNA and gene specific primers capable of binding to MYC (Thyroid, 11:2:147-152, 2001), EGFR (Clin. Cancer Res., 7:1850-1855, 2001) or FGFR-2 (Pediatr. Surg., 39:4:537-539, 2004) sequences.
  • EGFR Forward TGCGTCTCTTGCCGGAAT (SEQ ID NO: 1)
  • EGFR Reverse GGCTCACCCTCCAGAAGCTT SEQ ID NO: 2
  • Taqman 6FAM5'-
  • ACGCATTCCCTGCCTCGGCTG-3 'TAMRA SEQ ID NO: 3
  • FGFR-2 Forward CAATGTGACGGAGATGGATG SEQ ID NO: 4
  • FGFR-2 Reverse GATGACTGTCACCACCATGC SEQ ID NO: 5
  • MYC Forward CAAACCTCCTCACAGCCCACT SEQ ID NO: 6
  • MYC Reverse TTCGCCTCTTGACATTCTCCTC SEQ ID NO: 7
  • Other gene specific primers can be used as well, such as primers corresponding to Affymetrix probe set sequences 210984_X_AT, 202431_S_AT, and 211401_S_AT.
  • Amplification products can then be visualized and quantified following electrophoresis.
  • An increase in MYC, EGFR or FGFR-2 expression by at least 2- fold (such as at least 4-fold, at least 5-fold, or at least 10-fold as compared to a reference value or range of values (e.g., expected expression levels or range of levels of each marker in a chemosensitive tumor) indicates that the subject has a chemoresistant tumor.
  • a protein expression product can be detected by standard Western blot or immunoassay techniques that are known to one of skill in the art.
  • biopsy tissue samples are collected during the diagnostic endoscopy procedure, and immediately frozen in liquid nitrogen.
  • Serial cryosections of the biopsy samples embedded in OCT can be made, until a representative section containing the majority of the embedded tissue is identified.
  • the corresponding tumor-rich area is manually dissected out of the remaining OCT block in a cryostat, with care taken to avoid contamination of non-neoplastic cells in the tumor samples.
  • Immunohistochemistry can then be performed using paraffin-embedded formalin- fixed biopsy samples.
  • Primary antibodies used for immunohistochemistry are sc- 003 for EGFR, sc-122 for FGFR2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and 9E10 for MYC (BD Biosciences, San Jose, CA).
  • Various titers of primary antibodies can be used.
  • titers of primary antibodies can be 1 : 100 (overnight hybridization), 1:50 (30 minute hybridization), and 1:500 (overnight hybridization) for EGFR, FGFR2, and MYC, respectively.
  • Secondary antibody titer can be 1:100.
  • Detecting an at least 2-fold increase (such as at least 4-fold, at least 5- fold, or at least 10-fold increase) in EGFR, FGFR-2 and MYC expression as compared to a reference value or range of values (e.g., expected expression levels or range of levels of each marker in a chemosensitive tumor) indicates that the subject has a chemoresistant tumor.
  • a reference value or range of values e.g., expected expression levels or range of levels of each marker in a chemosensitive tumor
  • This example describes methods used to identify a gene signature that can be used to predict a subject's response to chemotherapy, such as acquired resistance to CF chemotherapy.
  • This algorithm computes a paired z-test for each gene using the RMA- summarized log- intensities for Affymetrix U133A arrays, and the proportion of the random permutations that gives as many genes significant at various levels (0.001, 0.005, 0.01, and 0.05) as are found in comparing the true class labels.
  • a P value was computed for each gene for the correlation of expression level vs. survival time using a proportional hazards model.
  • a summary statistic was computed that summarizes these P values over the gene set; the summary statistic was average 1Og(P) for LS summary of how the P values differ from a uniform distribution for LS (Pavlidis et al, Neurochem. Res. 29:1213-1222, 2004).
  • the summary statistic was related to the distribution of the summary statistics for random samples of 468 genes, sampled from those represented on the array. 100,000 random gene sets were sampled to compute this distribution.
  • the LS P value was the proportion of random sets of 468 genes with smaller average summary statistics than the LS summaries computed for the real data.
  • This approach can be used for a variety of types of correlations of gene expression level to phenotype; the way the gene specific P values are computed was determined by the nature of the phenotype ⁇ e.g. survival time or binary indicators). The LS P value less than 0.005 was considered significant.
  • This example describes methods used to identify 50 chemotherapy sensitivity-related molecules that can be used to predict a subject's response to chemotherapy, such as the chemoresponsiveness of a metastatic gastric cancer response to CF chemotherapy.
  • Target genes for MYC were selected based on the work by Ben- Porath et al. ⁇ Nature Genet. 40:466-507, 2008), in which several gene sets associated with embryonic stem cell identity were compiled for gene set comparison analyses.
  • MYC target genes were originally identified by a chromatin immunoprecipitation (ChIP) array study in Burkitt's lymphoma (Li et al, Proc. Natl. Acad. ScL USA 100:8164-8169, 2003).
  • ChiIP chromatin immunoprecipitation
  • Entrez IDs of MYC target genes used by Ben-Porath et al. were mapped to 775 probe set IDs on the HG-U133A array (available on the World Wide Web at NetAffx.com).
  • Transcriptional factor analysis was performed to look for the enrichment of transcriptional factor targets in the gene signatures (BRB-Array Tools). All genes in this analysis algorithm have been catalogued to transcriptional factor responsive categories based upon experimentally-verified transcription factor responsiveness. Transcription factor- binding curation information in the Transcriptional Regulatory Element Database (TRED) (Zhao et al., Nucl. Acids Res. 33:D103-107, 2005) was used to eliminate targets without any experimental verification. Development of the 50- gene predictive index.
  • TRED Transcriptional Regulatory Element Database
  • a genomic predictor (referred to as "50-gene predictive index") was developed, which was the weighted linear combination of log signal values of 50 unique genes represented by these 60 probe sets.
  • the univariate /-statistics for comparing the classes were used as the weights.
  • BRB -Array Tools (the class prediction) was used to calculate the z-value of each gene.
  • TRED MYC_T00140 The experimentally- validated MYC gene target gene set (TRED MYC_T00140) and MYC target genes identified by a chromatin immunoprecipitation (ChIP) array study (Li et al, Proc. Natl. Acad. ScL USA 100:8164-8169, 2003) were found to be significantly enriched in the acquired resistance signature described in Example 4 (LS P values ⁇ 10 "5 and 0.00002). Of 468 upregulated genes in the acquired resistance signature, 50 genes (60 probe sets) were identified as MYC target genes (Table 7), based upon BRB-Array Tools transcriptional factor analysis or a MYC ChIP study dataset (FIG. 4A).
  • This example describes methods used to identify 72 chemotherapy sensitivity-related molecules that can be used to predict a subject's response to chemotherapy, such as the chemoresponsiveness of a metastatic gastric cancer response to CF chemotherapy.
  • 72 unique genes were members of at least one of four published ES cell-related gene sets (excluding proliferation genes; Ben-Porath et al, Nature Genet. 40:499-507, 2008; Hu et al, BMC Genomics 7:96, 2006), the experimentally-validated MYC transcriptional factor target gene set
  • the univariate /-statistics for comparing the classes were used as the weights.
  • BRB -Array Tools (the class prediction) was used to calculate the t- value of each gene.
  • the predictive power of the 72-gene predictive index was tested for time to progression and survival by Cox proportional hazards model.
  • ES cells are highly proliferative in vitro, while stem cells are quiescent in vivo, the ES expression set was modified to exclude genes listed in the "proliferation" category of Gene Ontology and the proliferation cluster of breast cancer.
  • the overlap between this amended ES expression set ("ES set without proliferation genes") and the acquired resistance signature was still statistically significant (LS P value 4.0 x 10 ⁇ 3 ).
  • target genes of MYC and S OX2 which are known to be overexpressed in ES cells, were enriched in the acquired resistance signature (Table 9).
  • genes represented in the ES cell signature represent a core set of genes associated with in the acquired resistance was tested. From 633 genes in the acquired resistance signature (P ⁇ 0.01), 72 unique genes were extracted that belong to ES cell-related gene sets (either ES set without proliferation genes or MYC/SOX2-target genes) and were upregulated in the chemoresistant state at P ⁇ 0.01 (designated as the "72-gene acquired resistance signature") (Table 10). Genes downregulated in the chemoresistance signature were intentionally left out of the predictive model, because ES set without proliferation genes and MYC/SOX2- target genes are known to be overexpressed in ES cells.
  • the target genes of SUZ12, EED, and H3K27 which are known to be under-expressed in ES cells, were not enriched in the acquired resistance signature.
  • the 50 MYC target genes identified in the acquired resistance signature (Table 7, Example 5) are also included in the 72-gene ES acquired resistance signature (Table 10).
  • Hierarchical clustering was performed with the 101 pretreatment samples from a separate cohort of CF-treated patients who were not re -biopsied. Two main clusters were generated with patients in the "high expression” cluster exhibiting more rapid disease progression and poorer survival than patients in the cluster with "lower expression” (Log-rank P values, 0.028 and 0.028) (FIGS 5A and B).
  • the multivariable Cox regression analysis demonstrated that the 72-gene predictive index, as a continuous variable, was an independent predictor for time to progression and overall survival, after adjustment for age, sex, and performance status (Table 11).
  • Weighted linear combination of log signal values of 72-gene acquired resistance signature The univariate t-statistics for comparing the acquired chemoresistant state with the pretreatment state were used as the weights.
  • This example describes exemplary methods that can be used to test whether altering expression of the chemosensitivity-associated molecules described herein modulates the sensitivity of tumor cells to CF chemotherapy.
  • Tumor cells such as a gastric cancer cell line or primary gastric cancer cells are treated with a composition including one or more agents that decrease the biological activity of EGFR, FGFR-2, and/or MYC (for example, siRNA, antibodies, or small molecule inhibitors).
  • a composition including one or more agents that decrease the biological activity of EGFR, FGFR-2, and/or MYC for example, siRNA, antibodies, or small molecule inhibitors.
  • Appropriate dosages can be determined by one of skill in the art, for example, by testing a range of doses and measuring expression or biological activity of EGFR, FGFR-2, and/or MYC.
  • the cells are treated with CF for a defined period of time (for example, about 1 to 5 days).
  • Cell survival is measured, for example, using a trypan blue exclusion test. Decreased cell survival compared to a control (for example, cells treated with the agents that decrease biological activity of EGFR, FGFR-2, and/or MYC, but without CF) indicates that the cells have increased sensitivity to CF chemotherapy.
  • tumor cells such as a gastric cancer cell line
  • the treated tumor cells are implanted in an immune-compromised mouse and tumor is allowed to form. The mouse is then treated with CF. Growth of the tumor (for example, tumor volume, tumor cell number, tumor metastasis) is monitored.
  • a decrease in tumor growth for example, compared to a mouse implanted with tumor cells not treated with a composition including one or more agents that decrease the biological activity of EGFR, FGFR-2, and/or MYC and treated with CF, indicates that the tumor cells have increased sensitivity to CF chemotherapy.
  • Example 8 Predicting Chemotherapy Sensitivity
  • a subject's responsiveness to chemotherapy can be predicted prior to providing chemotherapy by detecting increased expression of one or more ES markers or MYC targets in a sample obtained from the subject with an epithelial cancer, such as metastatic gastric cancer.
  • the presence of increased expression of one or more ES markers (such as those provided in Table 10) or MYC targets (such as those provided in Table 7) indicates that the cancer has a decreased sensitivity to chemotherapy treatment.
  • the expression product can be RNA or protein.
  • biopsy tissue samples can be collected during an endoscopy procedure, and immediately frozen in liquid nitrogen.
  • An RNA expression product can be detected by PCR according to methods known to those of skill in the art.
  • a tissue sample is obtained from a subject identified to have gastric cancer (such as prior to any chemotherapy treatment, or after the development of chemotherapy resistance).
  • At least one microgram of RNA is then isolated from the samples that contain at least 50% tumor cells by using TRI Reagent (Invitrogen, Carlsbad, CA).
  • cDNA can then be synthesized from the isolated RNA.
  • PCR is performed in the presence of PCR reagents, cDNA synthesized from the isolated RNA and gene specific primers capable of binding to ES marker sequences (such as sequences of ES markers provided in Table 10) and/or MYC target sequences (such as sequences of MYC targets provided in Table 7).
  • ES marker sequences such as sequences of ES markers provided in Table 10
  • MYC target sequences such as sequences of MYC targets provided in Table 7
  • Amplification products can then be visualized and quantified following electrophoresis.
  • An increase in ES marker or MYC target expression by at least 2- fold (such as at least 4-fold, at least 5-fold, or at least 10-fold) as compared to a reference value or range of values (e.g., expected expression levels or range of levels of each marker in a chemosensitive tumor) indicates that the subject has a chemoresistant tumor.
  • a protein expression product can be detected by standard Western blot or immunoassay techniques that are known to one of skill in the art. In a particular example, biopsy tissue samples are collected during the endoscopy procedure, and immediately frozen in liquid nitrogen.
  • titers of primary antibodies can be 1:100 (overnight hybridization), 1:50 (30 minute hybridization), and 1:500 (overnight hybridization). Secondary antibody titer can be 1:100.
  • Detecting an at least 2-fold increase (such as at least 4-fold, at least 5-fold, or at least 10-fold increase) in ES marker or MYC target expression as compared to a reference value or range of values (e.g., expected expression levels or range of levels of each marker in a chemosensitive tumor) indicates that the subject has a chemoresistant tumor.
  • This example describes methods that can be used to significantly reduce chemoresistance in a subject with an epithelial cancer, such as metastatic gastric cancer.
  • chemoresistance can be reduced or inhibited by administering a therapeutically effective amount of a composition including one or more agents that decrease the biological activity of EGFR, FGFR- 2, MYC, and/or one or more ES markers or MYC targets that are up-regulated in chemoresistant gastric tumors, thereby reducing or inhibiting chemoresistance in the subject.
  • a subject who has been diagnosed with gastric cancer is identified and then determined if chemoresistant by any of the methods disclosed herein.
  • a therapeutic effective dose of the composition including the one or more agents that decrease the biological activity of EGFR, FGFR-2, MYC, and/or one or more ES markers or MYC targets is administered to the subject.
  • a therapeutic effective dose of a composition including one or more agents that decrease the biological activity of EGFR, FGFR-2, MYC, and/or one or more ES markers or MYC targets is administered to the subject to inhibit chemoresistance.
  • the composition includes one or more siRNAs.
  • the composition includes one or more antibodies.
  • a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (e.g. , chemoresistance) in a subject without causing a substantial cytotoxic effect in the subject.
  • siRNAs are administered according to the teachings of Soutschek et al. (Nature Vol. 432: 173-178, 2004) or Karpilow et al. (Pharma Genomics 32-40, 2004) both of which are herein incorporated by reference in their entireties.
  • siRNAs specific from EGFR, FGFR, and MYC are incorporated into neutral liposomes, such as DOPC, and injected intraperitoneal or intravenously.
  • a siRNA is administered at 150 ⁇ g/kg twice weekly for 2 to 3 weeks.
  • one or more siRNAs for each of EGFR, FGFR and MYC is administered at 150 ⁇ g/kg twice weekly for 2 to 3 weeks.
  • naked antibodies for EGFR, FGFR, MYC, one or more ES markers or MYC targets, or combination thereof are administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks depending upon the chemoresistance.
  • the antibodies are administered continuously.
  • antibodies or antibody fragments conjugated to cytotoxic agents (immunotoxins) are administered at 50 ⁇ g per kg given twice a week for 2 to 3 weeks.
  • the subject is administered the therapeutic composition that a binding agent specific for one or more of the disclosed chemotherapy sensitivity-related molecules daily for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.
  • subject will be administered CF therapy concurrently or following treatment with either siRNAs or antibodies specific for EGFR, FGFR-2, MYC and/or one or more ES markers or MYC targets.
  • Subjects can receive cisplatin 60 mg/m 2 IV on day 1 and fluorouracil 1,000 mg/m 2 IV on days 1-5 of a 3- week schedule either concurrently with receiving the above detailed siRNA and antibody treatments or following such treatments.
  • Subjects can continue therapy indefinitely until they experienced unacceptable toxicities or progressive disease is documented.
  • Subjects will be monitored by methods known to those skilled in the art to determine gastric tumor responsiveness to the siRNA or antibody treatment.
  • the subject will be monitored by non invasive techniques such as CT or MRI imaging to assess tumor response.
  • additional agents can be administered, such as antineoplastic agents in combination with or following treatment with the siRNA or antibodies.

Abstract

La présente invention concerne des signatures génétiques décrivant des profils génétiques. Les signatures génétiques peuvent prédire si un sujet atteint d'un cancer, tel qu'un cancer de l'estomac, sera chimiorésistant ou chimiosensible à une chimiothérapie à base de cisplatine et de fluorouracile. L'invention décrit ainsi des méthodes permettant de déterminer si un sujet atteint d'un cancer sera sensible à un traitement avec un agent chimiothérapeutique. L'invention décrit des méthodes permettant d'augmenter la sensibilité à l'agent chimiothérapeutique si EGFR, FGFR-2 et MYC sont augmentés, cette augmentation indiquant que le cancer présente une sensibilité diminuée à un agent chimiothérapeutique. L'invention décrit également des méthodes permettant d'augmenter la sensibilité à l'agent chimiothérapeutique si un ou plusieurs marqueurs de cellules souches embryonnaires ou une ou plusieurs cibles MYC sont augmentés, cette augmentation indiquant que le cancer présente une sensibilité diminuée à un agent chimiothérapeutique.
PCT/US2009/059423 2008-10-03 2009-10-02 Prédicteurs de chimiorésistance par expression génique WO2010040083A2 (fr)

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US8481688B2 (en) 2010-05-11 2013-07-09 Aveo Pharmaceuticals, Inc. Anti-FGFR2 antibodies
US10378066B2 (en) 2010-09-15 2019-08-13 Almac Diagnostic Services Limited Molecular diagnostic test for cancer
US10214777B2 (en) 2010-09-15 2019-02-26 Almac Diagnostics Limited Molecular diagnostic test for cancer
US9540698B2 (en) 2011-11-17 2017-01-10 Genedia Biotech Co., Ltd. MIR-193A-3P and associated genes predict tumorigenesis and chemotherapy outcomes
WO2013071502A1 (fr) * 2011-11-17 2013-05-23 Genedia Biotech Co Ltd Gène mir-193a-3p et gènes associés prédisant la tumorigenèse et les résultats d'une chimiothérapie
US10161008B2 (en) 2011-11-17 2018-12-25 Genedia Biotech Co., Ltd. miR-193a-3p and associated genes predict tumorigenesis and chemotherapy outcomes
US9127320B2 (en) 2011-11-17 2015-09-08 Genedia Biotech Co., Ltd. miR-193a-3p and associated genes predict tumorigenesis and chemotherapy outcomes
WO2014018683A3 (fr) * 2012-07-24 2014-04-03 Cedars-Sinai Medical Center Nouvelle méthode de détection de la résistance à la chimiothérapie chez des patients atteints d'un cancer du poumon
CN104619343A (zh) * 2012-07-24 2015-05-13 雪松-西奈医学中心 一种检测肺癌患者化疗抗性的新方法
EP2695950A1 (fr) * 2012-08-10 2014-02-12 Blackfield AG Marqueurs pour la réactivité à un inhibiteur du récepteur du facteur de croissance des fibroblastes
CN105874080A (zh) * 2013-09-09 2016-08-17 阿尔玛克诊断有限公司 用于食道癌的分子诊断测试
WO2015033172A1 (fr) * 2013-09-09 2015-03-12 Almac Diagnostics Limited Test de diagnostic moléculaire du cancer de l'œsophage
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CN107254546A (zh) * 2017-08-16 2017-10-17 复旦大学附属华山医院 一种与乳腺癌新辅助化疗疗效相关的snp标志物及其应用
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