WO2010002955A2 - Biomarqueurs de souchitude et procédés d’utilisation - Google Patents

Biomarqueurs de souchitude et procédés d’utilisation Download PDF

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WO2010002955A2
WO2010002955A2 PCT/US2009/049343 US2009049343W WO2010002955A2 WO 2010002955 A2 WO2010002955 A2 WO 2010002955A2 US 2009049343 W US2009049343 W US 2009049343W WO 2010002955 A2 WO2010002955 A2 WO 2010002955A2
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genes
expression
sternness
sternness signature
cell
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PCT/US2009/049343
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English (en)
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WO2010002955A3 (fr
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Xudong Dai
Christopher G. Winter
Hongyue Dai
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Merck & Co., Inc.
Rosetta Inpharmatics Llc
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Publication of WO2010002955A2 publication Critical patent/WO2010002955A2/fr
Publication of WO2010002955A3 publication Critical patent/WO2010002955A3/fr

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    • 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
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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

Definitions

  • One aspect of the invention generally relates to use of selected gene expression biomarkers (sternness signature) to classify cells based upon the similarity to a threshold value of the sternness signature.
  • Other aspects of the invention are generally related to multiple uses of the sternness signature evaluate or compare agents that modify gene and/or protein activity and to predict subject response to cancer therapy.
  • sequence listing of the present application is submitted electronically via EFS- Web as an ASCII formatted sequence listing with a file name of "ROSONCOOOOl -S EQLIST- 18JUN2009.TXT", creation date of June 18, 2009 and a size of 203KB.
  • This sequence listing submitted via EF S -Web is part of the specification and is herein incorporated by reference in its entirety.
  • the classification of patient samples is a important aspect of cancer diagnosis and treatment.
  • the association of a patient's response to drug treatment with molecular and genetic markers can open up new opportunities for drug development in non- responding patients, or distinguish a drug's indication among other treatment choices because of higher confidence in the efficacy.
  • the pre-selection of patients who are likely to respond well to a medicine, drug, or combination therapy may reduce the number of patients needed in a clinical study or accelerate the time needed to complete a clinical development program (M. Cockett et al, 2000, Current Opinion in Biotechnology, 11 :602 ⁇ 609).
  • FIGURE 1 shows the response of the 58 sternness signature genes to perturbations affecting stem cell-like properties in vitro and in vivo.
  • Panel A shows the expression levels of the sternness signature genes in cells transfected with increasing concentrations of Sox2 siRNA and Oct4 siRNA.
  • Panel B shows the expression levels of the sternness signature genes in mouse embryonic stem cells as compared to mouse embryonic fibroblasts.
  • Panel C shows the expression levels of the sternness signature genes in mouse embryonic fibroblast cells that were genetically engineered to overexpress four transcription factors (Oct4, Sox2, c-Myc and Klf4) that regulate important stem cell properties.
  • FIGURE 2 shows that the sternness signature genes are over expressed in PTEN deficient mouse intestine.
  • FIGURE 3 shows the association of the expression levels of the sternness genes with tumor pathological phenotypes and clinical outcomes in breast cancer patients.
  • Panel A shows the level of expression of the sternness signature genes in patients with basal like breast cancer.
  • Panel B shows the level of expression of the sternness signature genes in patients with estrogen receptor negative (ER) breast cancer.
  • FIGURE 4 shows that the probability of overall survival (Panel A) and probability of being metastasis free (Panel B) for more that five years are significantly associated with the level of expression of the sternness signature genes in -330 breast cancer patients.
  • FIGURE 5 shows the association of the expression of the sternness signature genes with tumor progression in CML patients.
  • Panel A shows expression levels of the sternness signature genes patients with CML at various phases of the disease.
  • FIGURE 6 Panel A shows the effects of gamma secretase inhibitor on expression of the sternness signature genes in T-ALL and breast tumor mouse models.
  • Panel B shows sternness signature scores compared to the ⁇ C50 for gamma secretase inhibition in T-ALL cell lines.
  • FIGURE 7 shows the effect of paclitaxel on expression of the sternness signature genes in a variety of cell lines.
  • oligonucleotide sequences that are complementary to one or more of the genes described herein refers to oligonucleotides that are capable of hybridizing under stringent conditions to at least part of the nucleotide sequence of said genes. Such hybridizable oligonucleotides will typically exhibit at least about 75% sequence identity at the nucleotide level to said genes, preferably about 80% or 85% sequence identity or more preferably about 90% or 95% or more sequence identity to said genes.
  • Bind(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
  • cancer means, any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including leukemias, for example, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS related cancers such as Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, and Metastatic brain cancers; cancers of the head and neck including various lymphomas such as mantle cell lymphoma, non-Hod
  • hybridizing specifically to refers to the binding, duplexing or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Biomarker means any gene, protein, or an EST derived from that gene, the expression or level of which changes between certain conditions. Where the expression of the gene correlates with a certain condition, the gene is a biomarker for that condition.
  • Biomarker-derived polynucleotides means the RNA transcribed from a biomarker gene, any cDNA or cRNA produced therefrom, and any nucleic acid derived therefrom, such as synthetic nucleic acid having a sequence derived from the gene corresponding to the biomarker gene.
  • a gene marker is "informative" for a condition, phenotype, genotype or clinical characteristic if the expression of the gene marker is correlated or anti-correlated with the condition, phenotype, genotype or clinical characteristic to a greater degree than would be expected by chance.
  • the term “gene” has its meaning as understood in the art.
  • gene may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences. It will further be appreciated that definitions of gene include references to nucleic acids that do not encode proteins but rather encode functional RNA molecules such as tRNAs. For clarity, the term gene generally refers to a portion of a nucleic acid that encodes a protein; the term may optionally encompass regulatory sequences. This definition is not intended to exclude application of the term “gene” to non-protein coding expression units but rather to clarify that, in most cases, the term as used in this document refers to a protein coding nucleic acid.
  • the gene includes regulatory sequences involved in transcription, or message production or composition.
  • the gene comprises transcribed sequences that encode for a protein, polypeptide or peptide.
  • an "isolated gene" may comprise transcribed nucleic acid(s), regulatory sequences, coding sequences, or the like, isolated substantially away from other such sequences, such as other naturally occurring genes, regulatory sequences, polypeptide or peptide encoding sequences, etc.
  • the term “gene” is used for simplicity to refer to a nucleic acid comprising a nucleotide sequence that is transcribed, and the complement thereof, hi particular embodiments, the transcribed nucleotide sequence comprises at least one functional protein, polypeptide and/or peptide encoding unit.
  • this functional term “gene” includes both genomic sequences, RNA or cDNA sequences, or smaller engineered nucleic acid segments, including nucleic acid segments of a non-transcribed part of a gene, including but not limited to the non- transcribed promoter or enhancer regions of a gene.
  • Smaller engineered gene nucleic acid segments may express, or may be adapted to express using nucleic acid manipulation technology, proteins, polypeptides, domains, peptides, fusion proteins, mutants and/or such like.
  • the sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences ("5'UTR").
  • the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' untranslated sequences, or (“3'UTR").
  • Mitotic inhibitor refers to a drug or agent which inhibits mitosis. Mitotic inhibitors may be divided into two classes. One class includes agents which modulate microtubule dynamics. These agents may bind reversibly to tubulin and prevent microtubule assembly and disassembly. The second class includes non-tubulin binding agents, which regulate mitotic events vicariously by interacting with specific intracellular targets, such as mitotic kinesins, kinases, separase, etc.
  • mitotic inhibitors include, but are not limited to: tubulin inhibitors (such as taxanes, epothilones, vinca alkaloids, corobretastatin, eleutherobines); kinesin inhibitors (such as Monastrol, enastron, enastrol, VS-83, sulfoquinovosylacylglycerols, ispinesib, adociasulfate-2); mitotic kinase inhibitors (such as PLKl inhibitors wortmannin, scytonemin, staurosporine, ON-019010, BI-2536; aurora kinase inhibitors VX-680, MLN-8054, PHA-680632, PHA-739358, A2D-1152. VX-528, MP-235, Hesperadin, ZM-447439); and separase inhibitors (reviewed in Ivachtchenko et al, 2007,
  • Signature refers to a set of one or more differentially expressed genes that are statistically significant and characteristic of the biological differences between two or more cell samples, e.g., normal and diseased cells, or cells exposed to an agent or not.
  • a signature may be expressed as a number of individual unique probes complementary to signature genes whose expression is detected when a cRNA product is used in microarray analysis or in a PCR reaction.
  • a signature may be exemplified by a particular set of biomarkers.
  • a “similarity value” is a number that represents the degree of similarity between two things being compared.
  • a similarity value may be a number that indicates the overall similarity between a cell sample expression profile using specific phenotype-related biomarkers and a control specific to that template (for instance, the similarity to a "deregulated growth factor signaling pathway" template, where the phenotype is deregulated growth factor signaling pathway status).
  • the similarity value may be expressed as a similarity metric, such as a correlation coefficient, or may simply be expressed as the expression level difference, or the aggregate of the expression level differences, between a cell sample expression profile and a baseline template.
  • the terms “measuring expression levels,” “obtaining expression level,” and “detecting an expression level” and the like includes methods that quantify a gene expression level of, for example, a transcript of a gene, or a protein encoded by a gene, as well as methods that determine whether a gene of interest is expressed at all.
  • an assay which provides a “yes” or “no” result without necessarily providing quantification, of an amount of expression is an assay that "measures expression” as that term is used herein.
  • a measured or obtained expression level may be expressed as any quantitative value, for example, a fold-change in expression, up or down, relative to a control gene or relative to the same gene in another sample, or a log ratio of expression, or any visual representation thereof, such as, for example, a "heatmap" where a color intensity is representative of the amount of gene expression detected.
  • the genes identified as being differentially expressed in tumor cells having growth factor signaling pathway deregulation may be used in a variety of nucleic acid or protein detection assays to detect or quantify the expression level of a gene or multiple genes in a given sample.
  • Exemplary methods for detecting the level of expression of a gene include, but are not limited to, Northern blotting, dot or slot blots, reporter gene matrix (see for example, US 5,569.588) nuclease protection, RT-PCR, microarray profiling, differential display, 2D gel electrophoresis, SELDI-TOF, ICAT, enzyme assay, antibody assay, and the like.
  • a “patient” can mean either a human or non-human animal, preferably a mammal.
  • subject as refers to an organism or to a cell sample, tissue sample or organ sample derived therefrom, including, for example, cultured cell lines, biopsy, blood sample, or fluid sample containing a cell.
  • the subject or sample derived therefrom comprises a plurality of cell types.
  • the sample includes, for example, a mixture of tumor and normal cells.
  • the sample comprises at least 10%, 15%, 20%, et seq., 90%, or 95% tumor cells.
  • the organism may be an animal, including but not limited to, an animal, such as a cow, a pig, a mouse, a rat, a chicken, a cat, a dog, etc., and is usually a mammal, such as a human.
  • pathway is intended to mean a set of system components involved in two or more sequential molecular interactions that result in the production of a product or activity.
  • a pathway can produce a variety of products or activities that can include, for example, intermolecular interactions, changes in expression of a nucleic acid or polypeptide, the formation or dissociation of a complex between two or more molecules, accumulation or destruction of a metabolic product, activation or deactivation of an enzyme or binding activity.
  • pathway includes a variety of pathway types, such as, for example, a biochemical pathway, a gene expression pathway, and a regulatory pathway.
  • a pathway can include a combination of these exemplary pathway types.
  • Notch signaling pathway refers to the set of genes and proteins that comprise an intracellular signaling pathway that is important for cell-cell communication, which involves gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life. Representative genes are listed in Table 1.
  • Notch pathway agent refers to an agent which modulates the Notch signaling pathway.
  • Molecular targets of such inhibitors may include ⁇ -secretase complex (i.e., PSENEN, PSENl, PSEN2, NCSTN and APHlA), and any of the Notch signaling pathway genes listed in Table 1.
  • An agent may be a small molecule, a siRNA, shRNA or naiRNA, an antibody, or any other molecule that targets and modulates the biological activity of one or more gene, or corresponding protein encoded by the gene, of the Notch signaling pathway.
  • Gamma secretase inhibitors include, but are not limited to, agents such as those disclosed in WO 01/90084, WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO 2004/039370, WO 2005/030731, WO 2005/014553, WO 2004/089911, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137, WO 2004/031139, WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO 02/47671). Additional GSIs are described by Lamer (2004, Expert Opin. Ther. Patents 14:1403-20), in particular pages 1410-11; and in WO 00/2476
  • sternness signature refers to one or more genetic markers, i.e., transcripts and/or proteins, whose expression level is associated with, one or more phenotypic characteristics of an embryonic stem cell or of a cell that is considered or has been engineered to be a model stem cell.
  • exemplary phenotypic characteristics of a stem cell include, but are not limited to, the capacity of self renewal, i.e., ability to undergo cell division and give rise to one or more daughter cell having the same stem cell characteristic, and pluripotency, i.e., cells having the capacity to differentiate into a plurality of different cell types.
  • An exemplary model stem cell includes, but are not limited to, genetically engineered mouse embryonic fibroblast (MEF) cells that over-produce OCT4, Sox2, c-Myc and Klf4 (MEFiPS) (Okita et aL, 2007 Nature 448:313-318).
  • An exemplary mouse embryonic stem cell line is the mouse RF8 cell line.
  • Exemplary human embryonic cell lines and sourcing vendors are listed at the National Institutes of Health Human Embryonic Stem Cell Registry and include: BGOl , BG02, BG03, SAOl, SA02JBS01, ES02, ES03, ES04, ES05, ES06, TE03, TE04, TE06, UCOl, UC06, WAOl, WA07, WA09, WA13 and WAl 4.
  • sternness signature genes is further described in Example 1 herein. Exemplary sternness signature genes are listed in Table 2.
  • treating in its various grammatical forms in relation to the present invention refers to preventing (i.e. chemoprevention), curing, reversing, attenuating, alleviating, minimizing, suppressing, or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g. bacteria or viruses), or other abnormal condition.
  • treatment may involve alleviating a symptom (i.e., not necessarily all the symptoms) of a disease of attenuating the progression of a disease.
  • Treatment of cancer refers to partially or totally inhibiting, delaying, or preventing the progression of cancer including cancer metastasis; inhibiting, delaying, or preventing the recurrence of cancer including cancer metastasis; or preventing the onset or development of cancer (chemoprevention) in a mammal, for example, a human, hi addition, the methods of the present invention may be practiced for the treatment of human patients with cancer. However, it is also likely that the methods would also be effective in the treatment of cancer in other mammals. As used herein, the term "therapeutically effective amount" is intended to qualify the amount of the treatment in a therapeutic regiment necessary to treat cancer.
  • the desired biological response is partial or total inhibition, delay, or prevention of the progression of cancer including cancer metastasis; inhibition, delay, or prevention of the recurrence of cancer including cancer metastasis; or the prevention of the onset of development of cancer (chemoprevention) in a mammal, for example, a human.
  • Dislaying or outputting a classification result, prediction result, or efficacy result means that the results of a gene expression based sample classification or prediction are communicated to a user using any medium, such as for example, orally, writing, visual display, etc., computer readable medium or computer system. It will be clear to one skilled in the art that outputting the result is not limited to outputting to a user or a linked external component(s), such as a computer system or computer memory, but may alternatively or additionally be outputting to internal components, such as any computer readable medium.
  • Computer readable media may include, but are not limited to hard drives, floppy disks, CD-ROMs, DVDs, DATs. Computer readable media does not include carrier waves or other wave forms for data transmission.
  • the various sample classification methods disclosed and claimed herein can, but need not be, computer-implemented, and that, for example, the displaying or outputting step can be done by, for example, by communicating to a person orally or in writing (e.g., in handwriting).
  • the invention provides a sternness signature (Table 2) and methods of use thereof.
  • a method is provided for classifying cell samples based on the degree to which the expression levels of genes within the sternness signature are similar to the expression levels of these genes in an embryonic stem cell, or a stem cell model system.
  • a method is provided for comparing agents that modulate gene and/or protein activity of a cell based on the degree to which the agents alter the expression levels of genes within the sternness signature.
  • a method is provided for predicting whether a subject with cancer will respond to treatment with a biologically active dose of a cancer therapeutic agent based on the degree to which the agents alter the expression levels of genes within the sternness signature.
  • a method is provided for predicting therapeutic efficacy of a cancer therapeutic agent based on the degree to which the agents alter the expression levels of genes within the sternness signature
  • Sternness Signature Biomarkers One aspect of the invention provides a set of 58 biomarkers whose expression is correlated with changes in the self-renewal and pluripotency characteristics of embryonic stem cells.
  • the expression levels of the genes represented in this "sternness signature" can be used evaluate, classify and/or predict the degree to which cells have self-renewal capability and pluripotency characteristics similar or dissimilar to embryonic stem cells.
  • Exemplary biomarkers identified as useful for classifying cell samples according to the sternness signature, predicting response of a cancer patient to an agent, or measuring pharmacodynamic effect of a therapeutic agent, are listed in Tables 2.
  • the gene transcript sequences corresponding to the sternness signature marker are set for as SEQ ID NOs: 1, 3, 5, 7, 10, 12, 14, 16, 18, 20, 22 5 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 45, 76, 78, 80, 82, 84, 86, 88, 90, 92, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, and 117.
  • subsets of the sternness signature may be used. For example, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, or, 57 or more, of the 58 markers set forth in Table 2 can be used to practice any combination of the 58 markers set
  • One aspect of the invention provides a method of using expression levels of the sternness signature genesets for classifying an isolated cell sample as having stem cell-like properties or not.
  • the method comprises: (a) calculating a measure of similarity between a first expression profile and a sternness signature template, the first expression profile comprising the expression levels of a plurality of genes in the isolated cell sample, the sternness signature template comprising expression levels of the plurality of genes that are average expression levels of the respective genes in a control cell sample having stem cell-like properties, the plurality of genes consisting of at least 5 genes for which markers are listed in Table 2; and
  • the method further includes the steps of:
  • the method further includes displaying; or outputting to a user interface device, a computer readable storage medium, or a local or remote computer system; the classification produced by said classifying step (b).
  • the degree of similarity between a gene expression profile obtained from a cellular sample and the sternness signature template profile can be determined using any method known in the art.
  • Dai et al describe a number of different ways of calculating gene expression templates and corresponding gene marker genesets useful in classifying breast cancer patients (US 7,171,311; WO2002103320; WO2005086891; WO2006015312; WO2006084272).
  • Linsley et al., (US 20030104426) and Radish et al., (US 20070154931) disclose gene markers genesets and methods of calculating gene expression template useful in classifying chronic myologenous leukemia patients.
  • the similarity is represented by a correlation coefficient between the sample profile and the template.
  • a correlation coefficient above a correlation threshold indicates high similarity, whereas a correlation coefficient below the threshold indicates low similarity.
  • the correlation threshold is set as 0.3, 0.4, 0.5 or 0.6.
  • similarity between a sample profile and a template is represented by a distance between the sample profile and the template.
  • a distance below a given value indicates high similarity, whereas a distance equal to or greater than the given value indicates low similarity.
  • the sternness signature template is calculated using an error-weighted average of the sternness signature gene expression levels.
  • the isolated cell sample is from a mammalian subject, such as for example, a rat, mouse, monkey, dog, pig, or human.
  • the isolated cell sample contains cancer cells, hi one such embodiment, if the tumor cell sample is classified as having a high similarity to the sternness signature template, then the subject from whom the cell sample was obtained is a candidate for treatment with a cancer therapeutic agent that down regulates expression of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30 of the 58 genes for which biomarkers are listed in Table 2.
  • the cancer therapeutic agent is a gamma secretase inhibitor compound.
  • the cell sample classification method is repeated a plurality of times using a cells sample exposed to a test agent and a corresponding control cell sample not exposed to the test agent, hi one embodiment, a high through-put PCR gene expression assays is used to screen at least 10, at least 100, at least 1000, or at least 10,000 different agents to identify agents that modulate the sternness signature genesets of the present invention. In one such embodiment, an siRNA library representing at least 10 percent of the genes of the human genome is screened to identify genes whose function modulates expression of the sternness signature genesets.
  • a siRNA library representative of at least 20 percent, at least 30 percent, at least 40, at least 50 percent, 60 percent, at least 70 percent, at least 80, or at least 90 percent of the genes of the human genome is screened, hi another embodiment the method is performed using small molecule libraries.
  • a plurality of different cell samples are compared and sorted based on a calculated sternness signature score for each cell sample.
  • the method comprises the steps of: a) obtaining an expression profile from a cell sample, said expression profile comprising expression levels of a plurality of genes in said sample of cells, said plurality of genes consisting of at least 5 genes for which markers are listed in Table 2; c) calculating a sternness signature score for said expression profile, wherein said sternness signature score is an average expression level calculated from the expression level obtained for each gene in said plurality of genes; d) repeating steps a) through c) for each of said plurality of different cell samples to obtain a plurality of sternness signature scores; and e) rank order listing said plurality of sternness signature scores by numeric value wherein said plurality of different cell samples are compared based on each calculated sternness signature score.
  • the above described method of comparing cell samples is useful in comparing a plurality of different cell samples such as, for example, cell samples obtained from different tumor biopsies from the same subject, or tumor biopsy sample obtained from a number of different subjects.
  • Such tumor samples can then be sorted using the sternness signature score, based on the degree to which the tumor cell samples resemble stem cells.
  • the subject is in need of treatment with a cancer therapeutic compound that causes the sternness signature score to become lower, i.e., more like the sternness score of non-cancerous tissue.
  • a second aspect of the invention relates to using changes in expression the sternness signature genes to compare a plurality of agents.
  • the method comprises the steps of: a) contacting a biologically effective amount of an agent with a sample of isolated mammalian cells; b) obtaining an expression profile, the expression profile comprising expression levels of a plurality of genes in the sample of cells, the plurality of genes consisting of at least 5 genes for which markers are listed in Table 2; c) calculating a sternness signature score for the agent, wherein the sternness signature score is an average expression level calculated from the expression level obtained for each gene in the plurality of genes; d) repeating steps a) through c) for each of the plurality of agents to obtain a plurality of sternness signature scores; and e) listing the plurality of sternness signature scores by numeric value, wherein the plurality of agents are compared based on the value of the sternness signature score.
  • the method additional includes the step of generating said expression profile by hybridization of nucleic acids derived from said sample contacted with said agent compared to nucleic acids derived from a second sample of isolated mammalian cells not contacted with said agent.
  • a sternness signature score is calculated for said human cell line prior to contact with said agent.
  • the isolated cell sample is an embryonic stem cell line or a non-stem cell line engineered to overexpress a plurality of transcription factors that regulate stem cell-like properties.
  • the isolated cell sample is a mouse or human embryonic stem cell line.
  • the isolated cell sample is a cancer cell line.
  • a high through-put gene expression assay is used to screen at least 10, at least 100, at least 1000, or at least 10,000 different agents to identify agents that modulate the sternness signature genesets of the present invention.
  • an siRNA library representing at least 10 percent of the genes of the human genome is screened to identify genes whose function modulates expression of the sternness signature genesets.
  • a siRNA library representative of at least 20 percent, at least 30 percent, at least 40, at least 50 percent, 60 percent, at least 70 percent, at least 80, or at least 90 percent of the genes of the human genome is screened.
  • the method is performed using small molecule libraries.
  • a third aspect of the invention relates to using changes in expression the sternness signature genes to predict whether a mammalian subject with cancer will respond to treatment with a biologically active dose of a cancer therapeutic agent.
  • the method comprises: a) obtaining a first expression profile comprising expression levels of a plurality of genes in a first cell sample comprising cancer cells isolated from the subject prior to exposure or treatment of the subject with the cancer therapeutic agent, or alternatively, prior to a time in which the agent acts to change gene expression, for example, within about 1 hour, 2 hours, 3 hours or 4 hours of exposure of the subject to the cancer therapeutic agent, the plurality of genes consisting of at least 5 genes for which markers are listed in Table 2; b) obtaining a second expression profile comprising the expression levels of the plurality of genes in a second cell sample comprising cancer cells isolated from the subject after more than about 4 hours, 6 hours, 8 hours or 12 hours of exposure of the subject to the cancer therapeutic agent, or alternatively, sufficient time has elapsed to allow the agent to initiate changes in gene expression
  • the method includes a step whereby the prediction result produced from step (d) is displayed; or outputted to a user interface device, a computer readable storage medium, or a local or remote computer system.
  • the method includes the additional step of generating said the expression profile and the second expression profiles by hybridization of nucleic acids derived from the first and second cell samples compared to nucleic acids derived from a control cell sample.
  • a method for predicting the therapeutic efficacy of an agent for treatment of cancer comprising: a) contacting a biologically effective amount of the agent with a sample of cells comprising cancer cells isolated from a subject suspected of having cancer; b) obtaining a plurality of expression profiles from the contacted cells, each expression profile obtained from cells contacted with the agent for a plurality of different time periods, each of the expression profiles comprising expression levels of a plurality of genes in the sample of cells, the plurality of genes consisting of at least 5 genes for which markers are listed in Table 2; c) calculating a sternness signature score for each of the plurality of expression profiles, wherein the sternness signature score is an average expression level calculated from the expression level obtained for each gene in the plurality of genes; and d) comparing the sternness signature scores obtained from the plurality of expression profiles wherein a decrease in the sternness signature score between the expression profiles over time is indicative of the therapeutic efficacy of the agent.
  • the expression levels of the marker genes in a sample may be determined by any means known in the art.
  • the expression level may be determined by isolating and determining the level (i.e., amount) of nucleic acid transcribed from each marker gene. Alternatively, or additionally, the level of specific proteins encoded by a marker gene may be determined.
  • the level of expression of specific marker genes can be accomplished by determining the amount of mRNA, or polynucleotides derived therefrom, present in a sample. Any method for determining RNA levels can be used. For example, RNA is isolated from a sample and separated on an agarose gel. The separated RNA is then transferred to a solid support, such as a filter. Nucleic acid probes representing one or more markers are then hybridized to the filter by northern hybridization, and the amount of marker-derived RNA is determined. Such determination can be visual, or machine-aided, for example, by use of a densitometer. Another method of determining RNA levels is by use of a dot-blot or a slot-blot.
  • RNA, or nucleic acid derived therefrom, from a sample is labeled.
  • the RNA or nucleic acid derived therefrom is then hybridized to a filter containing oligonucleotides derived from one or more marker genes, wherein the oligonucleotides are placed upon the filter at discrete, easily- identifiable locations.
  • Hybridization, or lack thereof, of the labeled RNA to the filter-bound oligonucleotides is determined visually or by densitometer.
  • Polynucleotides can be labeled using a radiolabel or a fluorescent (i.e., visible) label.
  • RT-PCR reverse transcription followed by PCR
  • RT-PCR involves the PCR amplification of a reverse transcription product, and can be used, for example, to amplify very small amounts of any kind of RNA (e.g., mRNA, rRNA, tRNA).
  • RNA e.g., mRNA, rRNA, tRNA
  • RT-PCR is described, for example, in Chapters 6 and 8 of The Polymerase Chain Reaction, Mullis, K.B., et al., Eds., Birkhauser, 1994, the cited chapters of which publication are incorporated herein by reference.
  • ArrayPlateTM kits can be used to measure gene expression.
  • the ArrayPlateTM mRNA assay combines a nuclease protection assay with array detection. Cells in microplate wells are subjected to a nuclease protection assay. Cells are Iysed in the presence of probes that bind targeted mRNA species. Upon addition of Sl nuclease, excess probes and unhybridized mRNA are degraded, so that only mRNA:probe duplexes remain. Alkaline hydrolysis destroys the mRNA component of the duplexes, leaving probes intact.
  • ArrayPlatesTM contain a 16-element array at the bottom of each well. Each array element comprises a position-specific anchor oligonucleotide that remains the same from one assay to the next
  • the binding specificity of each of the 16 anchors is modified with an oligonucleotide, called a programming linker oligonucleotide, which is complementary at one end to an anchor and at the other end to a nuclease protection probe.
  • probes transferred from the culture plate are captured by immobilized programming linker.
  • Captured probes are labeled by hybridization with a detection linker oligonucleotide, which is in turn labeled with a detection conjugate that incorporates peroxidase.
  • the enzyme is supplied with a chemiluminescent substrate, and the enzyme-produced light is captured in a digital image. Light intensity at an array element is a measure of the amount of corresponding target mRNA present in the original cells.
  • the ArrayPlateTM technology is described in Martel, R.R., et al., Assay and Drug Development Technologies l(l):6l-7l, 2002, which publication is incorporated herein by reference.
  • DNA microarrays can be used to measure gene expression.
  • a DNA microarray also referred to as a DNA chip, is a microscopic array of DNA fragments, such as synthetic oligonucleotides, disposed in a defined pattern on a solid support, wherein they are amenable to analysis by standard hybridization methods (see Schena, BioEssays 18:427, 1996).
  • Exemplary microarrays and methods for their manufacture and use are set forth in T.R. Hughes et al., Nature Biotechnology /9:342-347, April 2001, which publication is incorporated herein by reference.
  • tissue array Kononen et al., 1998 Nat. Med 4:844-7.
  • tissue array multiple tissue samples are assessed on the same microarray.
  • the arrays allow in situ detection of RNA and protein levels; consecutive sections allow the analysis of multiple samples simultaneously.
  • any method known in the art may be utilized.
  • expression based on detection of RNA which hybridizes to the genes identified and disclosed herein is used. This is readily performed by any RNA detection or amplification, method known or recognized as equivalent in the art such as, but not limited to, reverse transcription-PCR, the methods disclosed in U.S. patent application Ser. No. 10/062,857 (filed on Oct. 25, 2001) as well as U.S. Provisional Patent Application 60/298,847 (filed Jun. 15, 2001) and 60/257,801 (filed Dec. 22, 2000), and methods to detect the presence, or absence, of RNA stabilizing or destabilizing sequences.
  • expression based on detection of DNA status may be used. Detection of the DNA of an identified gene as may be used for genes that have increased expression in correlation with a particular outcome. This may be readily performed by PCR based methods known in the art, including, but not limited to, Q-PCR. Conversely, detection of the DNA of an identified gene as amplified may be used for genes that have increased expression in correlation with a particular treatment outcome. This may be readily performed by PCR based, fluorescent in situ hybridization (FISH) and chromosome in situ hybridization (CISH) methods known in the art.
  • FISH fluorescent in situ hybridization
  • CISH chromosome in situ hybridization
  • a gene expression-based expression assay based on a small number of genes i.e., about 1 to 3000 genes
  • Quantitative real-time PCR measures PCR product accumulation through a dual-labeled fluorigenic probe.
  • a variety of normalization methods may be used, such as an internal competitor for each target sequence, a normalization gene contained within the sample, or a housekeeping gene.
  • Sufficient RNA for real time PCR can be isolated from low milligram quantities from a subject. Quantitative thermal cyclers may now be used with microfluidics cards preloaded with reagents making routine clinical use of multigene expression-based assays a realistic goal.
  • the gene markers of the sternness signature or a subset of genes selected from the sternness signature, which are assayed according to the present invention are typically in the form of total RNA or mRNA or reverse transcribed total RNA or mRNA.
  • General methods for total and mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997).
  • RNA isolation can also be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen (Valencia, CA) and Ambion (Austin, TX), according to the manufacturer's instructions.
  • TAQman quantitative real-time PCR can be performed using commercially available PCR reagents (Applied Biosystems, Foster City, CA) and equipment, such as ABI Prism 7900HT Sequence Detection System (Applied Biosystems) according the manufacturer's instructions.
  • the system consists of a thermocycler, laser, charge-coupled device (CCD), camera, and computer.
  • the system amplifies samples in a 96- well or 384-well format on a thermocycler.
  • laser-induced fluorescent signal is collected in real-time through fiber- optics cables for all 96 wells, and detected at the CCD.
  • the system includes software for running the instrument and for analyzing the data.
  • a real-time PCR TAQman assay can be used to make gene expression measurements and perform the classification and sorting methods described herein.
  • oligonucleotide primers and probes that are complementary to or hybridize to the sternness signature markers listed in Table 2 may be selected based upon the marker transcript sequences set forth in the Sequence Listing.
  • polynucleotide microarrays are used to measure expression so that the expression status of each of the markers in one or more of the inventive genesets described herein, is assessed simultaneously.
  • the microarrays of the invention preferably comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or more of the sternness signature markers, or all of the markers, or any combination or subcombination of sternness signature markers.
  • Type I error means a false positive and “Type II error” means a false negative; in the example of prediction of therapeutic response to exposure to an agent, Type I error is the mis- characterization of an individual with a therapeutic response to the agent as having being a non- responder to treatment, and Type II error is the mis-characterization of an individual with no response to treatment with the agent as having a therapeutic response.
  • Polynucleotides capable of specifically or selectively binding to the mRNA transcripts encoding the polypeptide biomarkers of the invention are also contemplated.
  • oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA 5 synthetic RNA, or other combinations of naturally occurring or modified nucleotides which specifically and/or selectively hybridize to one or more of the RNA products of the biomarker of the invention are useful in accordance with the invention.
  • the oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA, synthetic RNA, or other combinations of naturally occurring or modified nucleotides oligonucleotides which both specifically and selectively hybridize to one or more of the RNA products of the biomarker of the invention are used.
  • the polynucleotide used to measure the RNA products of the invention can be used as nucleic acid members stably associated with a support to comprise an array according to one aspect of the invention.
  • the length of a nucleic acid member can range from 8 to 1000 nucleotides in length and are chosen so as to be specific for the RNA products of the sternness signature biomarkers of the invention. In one embodiment, these members are selective for the RNA products of the invention.
  • the nucleic acid members may be single or double stranded, and/or may be oligonucleotides or PCR fragments amplified from cDNA. Preferably oligonucleotides are approximately 20-30 nucleotides in length.
  • ESTs are preferably 100 to 600 nucleotides in length. It will be understood to a person skilled in the art that one can utilize portions of the expressed regions of the biomarkers of the invention as a probe on the array. More particularly oligonucleotides complementary to the genes of the invention and or cDNA or ESTs derived from the genes of the invention are useful. For oligonucleotide based arrays, the selection of oligonucleotides corresponding to the gene of interest which are useful as probes is well understood in the art. More particularly it is important to choose regions which will permit hybridization to the target nucleic acids. Factors such as the Tm of the oligonucleotide, the percent GC content, the degree of secondary structure and the length of nucleic acid are important factors. See for example U.S. Pat. No. 6,551,784.
  • the measuring of the expression of the RNA product of the invention can be done by using those polynucleotides which are specific and/or selective for the RNA products of the invention to quantitate the expression of the RNA product.
  • the polynucleotides which are specific and/or selective for the RNA products are probes or primers.
  • these polynucleotides are in the form of nucleic acid probes which can be spotted onto an array to measure RNA from the sample of an individual to be measured.
  • the polynucleotides which are specific and/or selective for the RNA products of the invention are used in the form of probes and primers in techniques such as quantitative real-time RT PCR, using for example S YBR®Green, or using TaqMan® or Molecular Beacon techniques, where the polynucleotides used are used in the form of a forward primer, a reverse primer, a TaqMan labelled probe or a Molecular Beacon labelled probe.
  • the nucleic acid derived from the sample cell(s) may be preferentially amplified by use of appropriate primers such that only the genes to be analyzed are amplified to reduce background signals from other genes expressed in the breast cell.
  • the nucleic acid from the sample may be globally amplified before hybridization to the immobilized polynucleotides.
  • RNA, or the cDNA counterpart thereof may be directly labeled and used, without amplification, by methods known in the art.
  • a "microarray” is a linear or two- dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support such as, but not limited to, glass, plastic, or synthetic membrane.
  • the density of the discrete regions on a microarray is determined by the total numbers of immobilized polynucleotides to be detected on the surface of a single solid phase support, preferably at least about 50/cm 2 , more preferably at least about 100/cm 2 , even more preferably at least about 500/cm 2 , but preferably below about 1,000/cm 2 .
  • the arrays contain less than about 500, about 1000, about 1500, about 2000, about 2500, or about 3000 immobilized polynucleotides in total.
  • a DNA microarray is an array of oligonucleotides or polynucleotides placed on a chip or other surfaces used to hybridize to amplified or cloned polynucleotides from a sample. Since the position of each particular group of primers in the array is known, the identities of a sample polynucleotides can be determined based on their binding to a particular position in the microarray. Determining gene expression levels may be accomplished utilizing microarrays.
  • the following steps may be involved: (a) obtaining an mRNA sample from a subject and preparing labeled nucleic acids therefrom (the "target nucleic acids” or “targets”); (b) contacting the target nucleic acids with an array under conditions sufficient for the target nucleic acids to bind to the corresponding probes on the array, for example, by hybridization or specific binding; (c) optional removal of unbound targets from the array; (d) detecting the bound targets, and (e) analyzing the results, for example, using computer based analysis methods.
  • “nucleic acid probes” or “probes” are nucleic acids attached to the array
  • target nucleic acids are nucleic acids that are hybridized to the array.
  • all or part of a disclosed sternness signature marker sequence may be amplified and detected by methods such as the polymerase chain reaction (PCR) and variations thereof, such as, but not limited to, quantitative PCR (Q-PCR), reverse transcription PCR (RT- PCR), and real-time PCR, optionally real-time RT-PCR.
  • PCR polymerase chain reaction
  • Q-PCR quantitative PCR
  • RT- PCR reverse transcription PCR
  • real-time PCR optionally real-time RT-PCR.
  • the newly synthesized nucleic acids are optionally labeled and may be detected directly or by hybridization to a polynucleotide of the invention.
  • the nucleic acid molecules may be labeled to permit detection of hybridization of the nucleic acid molecules to a microarray. That is, the probe may comprise a member of a signal producing system and thus, is detectable, either directly or through combined action with one or more additional members of a signal producing system.
  • the nucleic acids may be labeled with a fluorescently labeled dNTP (see, e.g., Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press San Diego, Calif.) , biotinylated dNTPs or rNTP followed by addition of labeled streptavidin, chemiluminescent labels, or isotopes.
  • labels include "molecular beacons" as described in Tyagi and Kramer (Nature Biotech. 14:303, 1996).
  • the newly synthesized nucleic acids may be contacted with polynucleotides (containing sequences) of the invention under conditions which allow for their hybridization.
  • Hybridization may be also be determined, for example, by plasmon resonance (see, e.g., Thiel, et al. Anal. Chem. 69:4948, 1997).
  • a plurality, e.g., 2 sets of target nucleic acids are labeled and used in one hybridization reaction ("multiplex" analysis).
  • one set of nucleic acids may correspond to RNA from one cell and another set of nucleic acids may correspond to RNA from another cell.
  • the plurality of sets of nucleic acids may be labeled with different labels, for example, different fluorescent labels (e.g., fluorescein and rhodamine) which have distinct emission spectra so that they can be distinguished.
  • the sets may then be mixed and hybridized simultaneously to one microarray (see, e.g., Shena, et al., Science 270:467-470, 1995).
  • a number of different microarray configurations and methods for their production are known to those of skill in the art and are disclosed in U. S. Pat.
  • an array of oligonucleotides may be synthesized on a solid support.
  • Exemplary solid supports include glass, plastics, polymers, metals, metalloids, ceramics, organics, etc.
  • chip masking technologies and photoprotective chemistry it is possible to generate ordered arrays of nucleic acid probes.
  • These arrays which are known, for example, as "DNA chips” or very large scale immobilized polymer arrays (“VLSIPS®” arrays), may include millions of defined probe regions on a substrate having an area of about 1 cm 2 to several cm 2 , thereby incorporating from a few to millions of probes (see, e.g., U.S. Pat. No. 5,631,734).
  • labeled nucleic acids may be contacted with the array under conditions sufficient for binding between the target nucleic acid and the probe on the array.
  • the hybridization conditions may be selected to provide for the desired level of hybridization specificity; that is, conditions sufficient for hybridization to occur between the labeled nucleic acids and probes on the microarray.
  • Hybridization may be carried out in conditions permitting essentially specific hybridization.
  • the length and GC content of the nucleic acid will determine the thermal melting point and thus, the hyridization conditions necessary for obtaining specific hybridization of the probe to the target nucleic acid. These factors are well known to a person of skill in the art, and may also be tested in assays.
  • An extensive guide to nucleic acid hybridization may be found in Tijssen, et al. (Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N. Y., (1993)).
  • the methods described above will result in the production of hybridization patterns of labeled target nucleic acids on the array surface.
  • the resultant hybridization patterns of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection selected based on the particular label of the target nucleic acid.
  • Representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement, light scattering, and the like.
  • One such method of detection utilizes an array scanner that is commercially available (Affymetrix, Santa Clara, Calif.), for example, the 417® Arrayer, the 418® Array Scanner, or the Agilent GeneArray® Scanner.
  • This scanner is controlled from a system computer with an interface and easy-to-use software tools. The output may be directly imported into or directly read by a variety of software applications. Exemplary scanning devices are described in, for example, U. S. Pat. Nos. 5,143,854 and 5,424,186.
  • Nucleic acid specimens may be obtained from a subject to be tested using either
  • a sampling means is said to be “invasive” if it involves the collection of nucleic acids from within the skin or organs of an animal (including murine, human, ovine, equine, bovine, porcine, canine, or feline animal).
  • invasive methods include, for example, blood collection, semen collection, needle biopsy, pleural aspiration, umbilical cord biopsy. Examples of such methods are discussed by Kim, et al., (J. Virol. 66:3879-3882, 1992); Biswas, et al., (Ann. NY Acad. Sci. 590:582-583, 1990); and Biswas, et al. , (J. Clin. Microbiol. 29:2228-2233, 1991).
  • a “non-invasive” sampling means is one in which the nucleic acid molecules are recovered from an internal or external surface of the animal.
  • Examples of such "non- invasive” sampling means include, for example, “swabbing,” collection of tears, saliva, urine, fecal material etc.
  • one or more cells from the subject to be tested are obtained and RNA is isolated from the cells.
  • a sample of cells is obtained from the subject. It is also possible to obtain a cell sample from a subject, and then to enrich the sample for a desired cell type. For example, cells may be isolated from other cells using a variety of techniques, such as isolation with an antibody binding to the an epitope on the cell surface of the desired cell type.
  • the desired cells are in a solid tissue
  • particular cells may be dissected, for example, by microdissection or by laser capture microdissection (LCM) (see, e.g., Bonner, et al., Science 278:1481, 1997; Emmert-Buck, et al., Science 274:998, 1996; Fend, et at, Am. J. Path. 154:61, 1999; and Murakami, et al., Kidney hit. 58:1346, 2000).
  • LCM laser capture microdissection
  • RNA may be extracted from tissue or cell samples by a variety of methods, for example, guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin, et al., Biochemistry 18:5294-5299, 1979).
  • RNA from single cells may be obtained as described in methods for preparing cDNA libraries from single cells (see, e.g., Dulac, Curr. Top. Dev. Biol. 36:245, 1998; Jena, et al., J. Immunol. Methods 190:199, 1996).
  • RNA sample can be further enriched for a particular species.
  • poly(A)+RNA may be isolated from an RNA sample.
  • the RNA population may be enriched for sequences of interest by primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template- directed in vitro transcription (see, e.g., Wang, et aL, Proc. Natl. Acad. Sci.
  • RNA, enriched or not in particular species or sequences may be further amplified by a variety of amplification methods including, for example, PCR; ligase chain reaction (LCR) (see, e. g. ? Wu and Wallace, Genomics 4:560, 1989; Landegren, et al., Science 241:1077, 1988); self- sustained sequence replication (SSR) (see, e.g., Guatelli, et al., Proc. Natl. Acad. Sci.
  • LCR ligase chain reaction
  • SSR self- sustained sequence replication
  • PCR technology Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, N. Y., N. Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif., 1990) ; Mattila, et al., Nucleic Acids Res.
  • RNA amplification and cDNA synthesis may also be conducted in cells in situ (see, e.g., Eberwine, et al. Proc. Natl. Acad. Sci. USA 89:3010, 1992).
  • the instant sternness signature genesets are used with therapeutic agents either singularly or in combination to identify and sort agents that directly or indirectly change gene expression levels of the sternness signature genes. In this way, an inventive method can be used to identify agents that increase or decrease the sternness signature genes.
  • the sternness signature genesets are used to evaluate and sort cancer therapeutic agents. Based upon the guidance and examples provided herein, it would also be apparent to a person of skill in the art that the sternness signature genesets can be used to evaluate, sort and compare any cancer therapeutic compounds and combination therapies thereof. Combinations of the presently disclosed methods with therapeutic, chemotherapeutic and anti-cancer agents are within the scope of the invention.
  • Such agents include the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, HTV protease inhibitors, reverse transcriptase inhibitors, inhibitors of cell proliferation and survival signaling, bisphosphonates, aromatase inhibitors, siRNA therapeutics, ⁇ -secretase inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and agents that interfere with cell cycle checkpoints.
  • RTKs receptor tyrosine kinases
  • Estrogen receptor modulators refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism.
  • Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LYl 17081, toremifene, fulvestrant, 4-[7-(2,2 ⁇ dimethyl-l-oxopropoxy-4-methyl-2-[4-[2-(l- ⁇ iperidinyl)ethoxy] phenyl] -2H- 1 -benzopyran-3-yl)-phenyl-2,2-dimethylpropanoate, 4,4'- dihydroxybenzophenone-2,4-dinitro ⁇ henyI-hydrazone, and SH646.
  • Androgen receptor modulators refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism.
  • Examples of androgen receptor modulators include finasteride and other 5 ⁇ -reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.
  • Retinoid receptor modulators refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism.
  • retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, ⁇ - difluoromethylornithine, ILX23-7553, trans-N-(4'-hydroxyphenyl) retinamide, and N-4- carboxyphenyl retinamide.
  • Cytotoxic/cytostatic agents refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signal transduction pathways, antimetabolites, biological response modifiers, hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topo ⁇ somerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors.
  • cytotoxic/cytostatic agents include, but are not limited to, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatm, irofalven, dexifosfamide, cis-aminedichloro(2- methyl-pyrid ⁇ ne)platinum, benzylguanine,, glufosfamide, GPXlOO, (trans, trans, trans)-bis-mu- (hexan
  • proteosome inhibitors include but are not limited to lactacystin and MLN- 341 (Velcade).
  • microtubule inhibitors/microtubule-stabilising agents include paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'-norvincaleukoblastine, docetaxol, rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPRl 09881, BMS 184476, vinflunine, cryptophycin, 2,3,4,5, 6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl ⁇ L-valyl-L-prolyl-L-proline-t- butylamide, TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237)
  • topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, ⁇ -ethoxypropionyl-3 ' ⁇ '-O-exo-benzylidene-chartreusin, 9-methoxy-N,N-dimethyl-5- nitropyrazolo[3,4 j5-kl]acridine-2-(6H) propanamine, 1 -ammo ⁇ 9-ethyl-5-fluoro-2,3-dihydro-9- hydroxy-4-methyl- 1 H, 12H-benzo[de]pyrano[3 ' ,4' :b,7] -indolizino [ 1 ,2b] quinoline- 10,13(9H,15H)dione, lurtotecan, 7-[2 ⁇ (N-isopropylamino)ethyl]-(20S)camptothecin ) BNP1350, BNPIl
  • inhibitors of mitotic kinesins are described in Publications WO03/039460, WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678, WO04/039774, WO03/079973, WO03/099211, WO03/105855, WO03/106417, WO04/037171, WO04/058148, WO04/058700, WO04/126699, WO05/018638, WO05/019206, WO05/019205, WO05/018547, WO05/017190,
  • inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLPl, inhibitors of CENP-E, inhibitors of MCAK and inhibitors of Rab6-KIFL.
  • histone deacetylase inhibitors include, but are not limited to, SAHA, TSA, oxamflatin, PXDlOl, MG98 and scriptaid. Further reference to other histone deacetylase inhibitors may be found in the following manuscript; Miller, T. A. et al. J. Med. Chem. 4 ⁇ (24):5097-5116 (2003).
  • “Inhibitors of kinases involved in mitotic progression” include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in particular inhibitors of PLK- 1), inhibitors of bub- 1 and inhibitors of bub-Rl.
  • PLK Polo-like kinases
  • An example of an "aurora kinase inhibitor” is VX-680.
  • Antiproliferative agents includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS 9 GEM231, and MX3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylidenecytidine, 2'- fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-(3,4- dichlorophenyl)urea
  • monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.
  • HMG-CoA reductase inhibitors refers to inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase.
  • HMG-CoA reductase inhibitors include but are not limited to lovastatin (MEVACOR®; see U.S. Patent Nos. 4,231,938, 4,294,926 and 4,319,039), simvastatin (ZOCOR®; see U.S. Patent Nos. 4,444,784, 4,820,850 and 4,916,239), pravastatin (PRAV ACHOL®; see U.S. Patent Nos.
  • HMG-CoA reductase inhibitors as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.
  • Prenyl-protein transferase inhibitor refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including farnesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase).
  • FPTase farnesyl-protein transferase
  • GGPTase-I geranylgeranyl-protein transferase type I
  • GGPTase-II geranylgeranyl-protein transferase type-II
  • prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Patent No. 5,420,245, U.S. Patent No. 5,523,430, U.S. Patent No. 5,532,359, U.S. Patent No. 5,510,510, U.S. Patent No. 5,589,485, U.S. Patent No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ.
  • Angiogenesis inhibitors refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism.
  • angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors FIt-I (VEGFRl) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon- ⁇ , interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal antiinflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol.
  • NSAIDs nonsteroidal antiinflammatories
  • NSAIDs nonsteroidal anti
  • steroidal antiinflammatories such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, ⁇ -O-chloroacetyl-carbony ⁇ -fumagillol, thalidomide, angiostatin, troponin-1, angiotensin ⁇ antagonists (see Fernandez et al., J Lab. Clin. Med.
  • agents that modulate or inhibit angiogenesis and may also be used in combination with the methods of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)).
  • agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost. 80:10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101 :329-354 (2001)).
  • TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filed August 8 5 2001) and 60/349,925 (filed January 18, 2002).
  • Agents that interfere with cell cycle checkpoints refer to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents.
  • agents include inhibitors of ATR, ATM, the CHKl, CHK2 and Wee-1 kinases and cdk and cdc kinase inhibitors and are specifically exemplified by 7- hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.
  • agents that interfere with receptor tyrosine kinases refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression.
  • agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met.
  • Further agents include inhibitors of RTKs as described by Bume-Jensen and Hunter, Nature, 41 1:355-365, 2001.
  • “Inhibitors of cell proliferation and survival signalling pathway” refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US 2005/43361, 60/734188, 60/652737, 60/670469), inhibitors of Raf kinase (for example BAY-43-9006 ),
  • NSAID's which are potent COX-2 inhibiting agents.
  • an NSAID is potent if it possesses an ICs 0 for the inhibition of COX-2 of l ⁇ M or less as measured by cell or micro soma! assays .
  • NSAID's which are selective COX-2 inhibitors are defined as those which possess a specificity for inhibiting COX-2 over COX-I of at least 100 fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-I evaluated by cell or microsomal assays.
  • Such compounds include, but are not limited to those disclosed in U.S. Patent 5,474,995, U.S. Patent 5,861,419, U.S. Patent 6,001,843, U.S. Patent 6,020,343, U.S. Patent 5,409,944, U.S. Patent 5,436,265, U.S. Patent 5,536,752, U.S.
  • Inhibitors of COX-2 that are particularly useful in the instant methods are: 3-phenyl-4-(4- (meraylsulfonyl)phenyl)-2-(5H)-furanone; and 5-chloro-3-(4-methylsulfonyI)phenyl-2-(2-methyl- 5-pyridinyl)pyridine; or a pharmaceutically acceptable salt thereof.
  • ARCOXIA® ARCOXIA®, BEXTRA® and CELEBREX® or a pharmaceutically acceptable salt thereof.
  • angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]- 1 -oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino- 1 -[[3 ,5-dichIoro-4-(4- chlorobenzoyl) phenyl] methyl] - 1 ⁇ - 1 ,2 ,3 -triazole-4-carboxamide 5 CM 101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl- bis [imino-N-methyl-4,2-pyrrolocarbonylimino [N-methy 1-4,2-pyrrol
  • integrated circuit blockers refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ⁇ gand to the ⁇ y ⁇ 3 integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ⁇ gand to the ⁇ v ⁇ 5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the ⁇ y ⁇ 3 integrin and the ⁇ v ⁇ 5 integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells.
  • the term also refers to antagonists of the ⁇ v ⁇ 6 > ctvps?
  • ⁇ l ⁇ l > ⁇ 2 ⁇ l > «-5 ⁇ l? ot ⁇ l and ⁇ 6 ⁇ 4 integrals.
  • the term also refers to antagonists of any combination of ⁇ v ⁇ 3, ot v ⁇ 5, ⁇ v ⁇ 6 > ⁇ v ⁇ 8 > ⁇ l Pl, «2 ⁇ l, ⁇ s ⁇ 1 , a ⁇ ⁇ and ⁇ 4 integrins.
  • tyrosine kinase inhibitors include N-(trifluoromethylphenyl)- 5-methylisoxazol-4-carboxamide, 3-[(2 ,4-dimethylpyrrol-5-yl)methylidenyl)mdoIin-2-one, 17- (allylamino)- 17-demethoxygeldanamycin, 4-(3 -chloro-4-fluorophenylamino)-7-methoxy-6- [3 -(4- morpholinyl)propoxyl]quinazol ⁇ ie, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolinamine, BIBX1382, 2,3,9,10,11 S 12-hexahydro-10-(hydroxymethyl)- 10-hydroxy-9- methyl-9, 12-e ⁇ oxy- 1 H-diindolo[ 1 ,2,3 -fg:3 ⁇ 2 ⁇ V ⁇ k
  • Combinations with compounds other than anti-cancer compounds are also encompassed in the instant methods.
  • combinations of the instantly claimed compounds with PPAR- ⁇ (i.e., PPAR-gamma) agonists and PPAR- ⁇ (i.e., PPAR-delta) agonists are useful in the treatment of certain malingnancies.
  • PPAR- ⁇ and PPAR- ⁇ are the nuclear peroxisome proliferator-activated receptors ⁇ and ⁇ .
  • the expression of PPAR- ⁇ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J Cardiovasc, Pharmacol 1998; 31:909-913; J. Biol. Chem. 1999;274:9116-9121 ; Invest. Ophthalmol Vis.
  • PPAR- ⁇ agonists and PPAR- ⁇ / ⁇ agonists include, but are not limited to, thiazolidinediones (such as DRF2725, CS-Ol 1, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NPOl 10, DRF4158, NN622, GE262570, PNU182716, DRF552926, 2-[(5,7- dipropyl ⁇ 3 -trifiuoromethyl- 1 ,2-benzisoxazol-6-yl)oxy] -2-methylpropionic acid (disclosed in USSN 09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluoroph
  • a method of the instant invention may also be useful for evaluating the use of gene therapy for the treatment of cancer.
  • Gene therapy can be used to deliver any tumor suppressing gene. Examples of such genes include, but are not limited to, p53, which can be delivered via recombinant virus-mediated gene transfer (see U.S. Patent No.
  • a uPA/uPAR antagonist (Adenovirus-Mediated Delivery of a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice," Gene Therapy, August 1998;5(8):1105-13), and interferon gamma (J Immunol 2000; 164:217-222).
  • the methods of the instant invention may also be used in combination with an inhibitor of inherent multidrug resistance (MDR), in particular MDR associated with high levels of expression of transporter proteins.
  • MDR inhibitors include inhibitors of p-glycoprotein (P- gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and PSC833 (valspodar).
  • Neurokinin- 1 receptor antagonists of use in conjunction with the present invention are fully described, for example, in U.S. Patent Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European Patent Publication Nos.
  • the neurokinin- 1 receptor antagonist for use in conjunction with the methods of the present invention is selected from: 2-(R)-(l-(R)-(3,5- bis(trifluoromethyl)phenyl)ethoxy)-3-(S) ⁇ (4-fluorophenyl)-4-(3-(5-oxo-lH,4H ⁇ l,2,4- triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Patent No. 5,719,147.
  • a method of the instant invention may also be useful for evaluating the treatment or prevention of anemia.
  • an anemia treatment agent is, for example, a continuous erythropoiesis receptor activator (such as epoetin alfa).
  • a method of the instant invention may also be useful for evaluating the treatment or prevention of cancer in combination with P450 inhibitors including: xenobiotics, qumidine, tyramine, ketoconazole, testosterone, quinine, methyrapone, caffeine, phenelzine, doxorubicin, ⁇ oleandomycin, cyclobenzaprine, erythromycin, cocaine, furafyline, cimetidine, dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem, terfenadine, verapamil, Cortisol, itraconazole, mibefradil, nefazodone and nelfinavir.
  • P450 inhibitors including: xenobiotics, qumidine, tyramine, ketoconazole, testosterone, quinine, methyrapone, caffeine, phenelzine, doxorubicin, ⁇ ole
  • a method of the instant invention may also be useful for evaluating the treatment or prevention of cancer in combination with Pgp and/or BCRP inhibitors including: cyclosporin A, PSC833, GF120918, cremophorEL, fumitremorgin C, Kol32, Kol34, Iressa, Imatnib mesylate, EKI-785, CIl 033 , novobiocin, diethylstilbestrol, tamoxifen, resperpine, VX-710, tryprostatin A, flavonoids, ritonavir, saquinavir, nelfinavir, omeprazole, quLnidine, verapamil, terfenadine, ketoconazole, nifidepine, FK506, amiodarone, XR9576, indinavir, amprenavir, Cortisol, testosterone, LY335979, OC 144-093, erythromycin, vincristine, digoxin and
  • a method of the instant invention may also be useful for evaluating the treatment or prevention of cancer, including bone cancer, in combination with bisphosphonates (understood to include bisphosphonates, diphosphonates, bisphosphonic acids and diphosphonic acids).
  • bisphosphonates include but are not limited to: etidronate (Didronel), pamidronate (Aredia), alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or cimadronate, clodronate, EB-1053, mmodronate, neridronate, piridronate and tiludronate including any and all pharmaceutically acceptable salts, derivatives, hydrates and mixtures thereof.
  • a method of the instant invention may also be useful for evaluating the treatment or prevention of breast cancer in combination with aromatase inhibitors.
  • aromatase inhibitors include but are not limited to: anastrozole, letrozole and exemestane.
  • a method of the instant invention may also be used to evaluate siRNA, shKNfA or miRNA cancer therapeutics.
  • a method of the instant invention may also be used in combination with ⁇ -secretase inhibitors and/or inhibitors of NOTCH signaling.
  • Such inhibitors include compounds described in WO 01/90084, WO 02/30912, WO 01/70677, WO 03/013506, WO 02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO 03/093253, WO 2004/039800, WO
  • 2004/039370 WO 2005/030731, WO 2005/014553, USSN 10/957,251, WO 2004/08991 1, WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/031137, WO 2004/031139, WO 2004/031138, WO 2004/101538, WO 2004/101539 and WO 02/47671 (including LY-450139).
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with PARP inhibitors.
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with carboplatin and taxol.
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with 5-FU and cisplatin.
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with capecitabine.
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with oxaliplatin.
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with cisplatin.
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with gemcitabine.
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with radiotherapy,
  • a method of the instant invention may also be useful for evaluating the treatment of cancer in combination with the following therapeutic agents: abarelix (Plenaxis depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®); Alemtuzumabb (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostme (Ethyol®); anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®); bevacuzimab (Avastin®); bexarotene capsules (Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); busulfan intravenous
  • the scope of the instant methods encompasses the use of agents in combination with a second compound selected from: an estrogen receptor modulator, an androgen receptor modulator, a retinoid receptor modulator, a cytotoxic/cytostatic agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis inhibitor, PPAR- ⁇ agonists, PPAR- ⁇ agonists, an inhibitor of inherent multidrug resistance, an anti-emetic agent, an agent useful in the treatment of anemia, an agent useful in the treatment of neutropenia, an immunologic- enhancing drug, an inhibitor of cell proliferation and survival signaling, a bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, ⁇ -secretase and/or NOTCH inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an agent that interferes with a cell
  • treating cancer refers to administration to a mammal afflicted with a cancerous condition and refers to an effect that alleviates the cancerous condition by killing the cancerous cells, but also to an effect that results in the inhibition of growth and/or metastasis of the cancer.
  • the angiogenesis inhibitor to be used as the second compound is selected from a tyrosine kinase inhibitor, an inhibitor of epidermal-derived growth factor, an inhibitor of fibroblast-derived growth factor, an inhibitor of platelet derived growth factor, an MMP (matrix metalloprotease) inhibitor, an integrin blocker, interferon- ⁇ , interleukin-12, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, o-O-chloroacetyl-carbony ⁇ -furnagillol, thalidomide, angiostatin, troponin- 1, or an antibody to VEGF.
  • the estrogen receptor modulator is tamoxifen or raloxifene.
  • kits comprising the sternness signature biomarker sets above.
  • the kit contains a microarray or PCR primer sets ready for hybridization to target polynucleotide molecules, plus software for the data analyses described above.
  • a Computer system comprises internal components linked to external components.
  • the internal components of a typical computer system include a processor element interconnected with a main memory.
  • the computer system can be an Intel 8086-, 80386-, 80486-, Pentium®, or
  • Pentium®-based processor with preferably 32 MB or more of main memory.
  • the external components may include mass storage.
  • This mass storage can be one or more hard disks (which are typically packaged together with the processor and memory). Such hard disks are preferably of 1 GB or greater storage capacity.
  • Other external components include a user interface device, which can be a monitor, together with an inputting device, which can be a "mouse", or other graphic input devices, and/or a keyboard.
  • a printing device can also be attached to the computer.
  • a computer system is also linked to network link, which can be part of an Ethernet link to other local computer systems, remote computer systems, or wide area communication networks, such as the Internet.
  • This network link allows the computer system to share data and processing tasks with other computer systems.
  • a software component comprises the operating system, which is responsible for managing computer system and its network interconnections.
  • This operating system can be, for example, of the Microsoft Windows® family, such as Windows 3.1, Windows 95, Windows 98, Windows 2000, or Windows NT.
  • the software component represents common languages and functions conveniently present on this system to assist programs implementing the methods specific to this invention. Many high or low level computer languages can be used to program the analytic methods of this invention. Instructions can be interpreted during run-time or compiled.
  • Preferred languages include C/C++, FORTRAN and JAVA.
  • the methods of this invention are programmed in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including some or all of the algorithms to be used, thereby freeing a user of the need to procedurally program individual equations or algorithms.
  • Such packages include Mathlab from Mathworks (Natick, Mass.), Mathematica® from Wolfram Research (Champaign, 111.), or S-Plus®D from Math Soft (Cambridge, Mass.).
  • the software component includes the analytic methods of the invention as programmed in a procedural language or symbolic package.
  • the software to be included with the kit comprises the data analysis methods of the invention as disclosed herein, hi particular, the software may include mathematical routines for biomarker discovery, including the calculation of correlation coefficients between clinical categories (i.e., growth factor signaling pathway regulation status) and biomarker expression.
  • the software may also include mathematical routines for calculating the correlation between sample biomarker expression and control biomarker expression, using array-generated fluorescence data, to determine the clinical classification of a sample.
  • a user first loads experimental data into the computer system. These data can be directly entered by the user from a monitor, keyboard, or from other computer systems linked by a network connection, or on removable storage media such as a CD-ROM, floppy disk (not illustrated), tape drive (not illustrated), ZIP® drive (not Illustrated) or through the network. Next the user causes execution of expression profile analysis software which performs the methods of the present invention.
  • a user first loads experimental data and/or databases into the computer system. This data is loaded into the memory from the storage media or from a remote computer, preferably from a dynamic geneset database system, through the network. Next the user causes execution of software that performs the steps of the present invention.
  • Candidate genes for a sternness biomarker geneset were identified by looking for genes whose transcripts were down-regulated in a set of experimental perturbations that disrupted the self-renewal and pluripotency programs in mouse ES cells ( Figure 1). For example, it is known that retinoic-acid and RNA interference (RNAi) targeting Nanog, Oct4, Sox2, Esrrb and other stem cell-relevant transcription factors may compromise the self-renewal program of stem cells in vitro and in vivo (Ivanova et al., 2006 Nature 442:533-38; Loh, et al., 2006 Nat Genet 38:431- 40).
  • RNAi retinoic-acid and RNA interference
  • RNA extracted from stable Dicer hypomorphic HCT-116, DLD-I, and RKO cells was profiled against RNA obtained from the respective parental cell lines.
  • Genes that are down-regulated by both Dicer-/- in one of three cancer cell lines and perturbation of transcription factors critical for self-renewal in ES cells were selected as gene expression markers for the sternness signature.
  • genes were selected as sternness signature genes if its gene expression ratio (untreated/treated) at log 10 scale of less than -0.03 in at least 5 out of 6 time points for any one of the 9 perturbations, including RA treatment and shRNA treatments targeted to individual transcription factors as reported by Okita et al, (2007 Nature 448:313-17).
  • Dicer-/- For genes down-regulated by Dicer-/-, first, the expression level from Dicer-deficient cell lines were compared to their wild-type parental cell line. Then, genes were selected whose expression ratio at log 10 scale were less than -0.03 and had error model based p-values of less than 0.01 in at least 3 out 4 profiles in one of the three cell lines.
  • sternness signature markers includes genes whose expression levels are correlated with the self-renewal and pluripotency characteristics of embryonic stem cells. Table 2 lists the sternness signature genes.
  • Figure 1 shows the extent of down regulation in gene expression of the 58 sternness signature genes observed in mouse ES cells (Cell line CCE) upon transfection with shRNA vectors for self-renewal regulators as reported by Ivanova et al. (Ivanova et aL, 2006 Nature 442:533-38) Briefly, as reported by Ivanova et al., shRNA vectors specific for Nanog, Oct4, Sox2, Esrrb, Tbx3, Tell, Mm.343880 and Dppa4 as well as reference vector HlP were transfected into ES cells for 48 hours. Resulting cells were re-plated at the density 0,3x10 6 cells per 10 cm dish.
  • GFP+ cells were FACS isolated from the cell culture and treated as day 0 time point. Cells after re-plating were maintained in the presence of LIF. GFP+ cells from each culture were selected by FACS every day for 7 consecutive days to obtain 8 day time course (d ⁇ -d7). Total RNA was purified from 0.2x10 6 cells using the TRIzol Reagent (Invitrogen). The gene expression data published by Ivanova et al, was obtained (Supplementary data from Nature) and analyzed to obtain the results displayed in Figure 1 , Panel A.
  • Panels B and C indicate that the regulation of the 58 sternness signature genes is associated with the activity of self-renewal and pluripotency in mouse embryonic fibroblast (MEF) before and after restoration of embryonic stem cell feature.
  • the experimental methods, cell lines, etc. were reported by Okita et al., (2007 Nature 448:313-17) and the gene expression data analysis results presented in Figure 1 uncomfortable panels B and C, were obtained from the Gene Expression Omnibus database, dataset identifier: GSE7841 (Table Sl). Using this dataset, the expression levels of the 58 sternness signature genes in embroyonic stem (ES) cells were compared with the corresponding expression levels measured in MEF cells (Panel B) and in Nanog iPS cells (Panel C).
  • mice embryonic fibroblasts with restored germBne pluripotency and self-renewal capability induced by overexpression of 4 transcription factors that are important to stem cell programs (Oct4, Sox2, c- Myc, and Klf4) ( Figure 1C). These cells are referred to as mouse embryonic fibroblast induced pluripotent stem (MEF iPS) cells.
  • MEF iPS mouse embryonic fibroblast induced pluripotent stem
  • the chance that the association observed above occurred at random was found to be low (P 2.6X10 ⁇ 10 ).
  • the overexpression of the four transcription factors is necessary to induce the MEF iPS cell from MEF, but not necessary for maintaining the germline stem cell pluripotency in MEF iPS.
  • the overexpression of the candidate sternness genes were likely to be driven by the restoration of sternness in MEF iPS.
  • Example 3 Response of sternness signature genes in an PTEN deficiency murine model.
  • Expression levels for the sternness signature genes were extracted from gene expression profiling data obtained from the Gene Expression Omnibus data base from intestinal PTEN- deficient murine model. These data were then analyzed to determine whether the expression levels of the sternness gene set correlate to the increases in stem cell populations that were observed in the PTEN models (He e al., 2007, Nature Genetics 39:189-98; GEO dataset identifier: GSE 6078).
  • Figure 2 shows the extent of overexpression of the sternness signature genes in PTEN deficient intestine cells verse intestinal cells having fully functional PTEN.
  • 22 sternness genes were up-regulated dramatically in all intestinal samples from the PTEN deficient mice.
  • the probability of observing 22 of 58 randomly selected genes all up-regulated was estimated by hypergeometric statistics to be less than 2.6X10 "6 .
  • Example 4 Association of sternness gene expression with tumor progression and clinical outcome in breast cancer patients.
  • Tumor stem cells have been suspected of being responsible for drug resistance, tumor initiation, and metastasis in cancer patients. Supporting evidence continues to emerge as the understanding of stem cells and tumorigenesis improves (Leedham, et al., 2005, J Cell MoI Med. 9: 11 -24; Akala and Clarke, 2006, Curr Opin Genet Dev. 16:496-501 ; Clarke and Fuller, 2006, Cell 124:1111-15; Dean et al., 2005, Nat Rev Cancer. 5:275-84; Yin and Li, 2006, J Clin Invest 116:1195-201; Li and Li, 2006, Trends Biochem Sci. 31:589-95; and Weissman, 2005, Novartis Found Symp.
  • Patients having a sternness signature score higher than the 40 percentile of all of the sternness signature scores among the 311 patients were defined as representing a group of patients with tumors that had a high sternness score, i.e., the tumors cells were classified as having high similarity to stem cells and high probability of having stem cell-like characteristics.
  • Patients having a sternness signature score lower than the 60 percentile of all of the sternness signature scores among the 311 patients were defined as representing a group of patients with tumors that had a low sternness signature score, i.e., the tumors cells were classified as having low similarity to stem cells and low probability of having stem cell like characteristics.
  • prognoses associated with the expression level of sternness genes were confirmed in all four additional breast cancer data sets (Miller et al., 2005 PNAS 102:13550-55 (GEO dataset identifier: GSE 3494); Pawitan et al., 2005 Breast Cancer Research 7:R953-R967 (GEO dataset identifier: GSE 1456); Wang et al., 2005 Lancet 365:671-79 (GEO dataset identifier: GSE 2034); Sotiriou et ai, 2006 J. Nat. Cancer Instit. 98:262-72 (GEO dataset identifier: GSE 2990) (data not shown).
  • Example 5 Association of sternness gene expression with tumor progression and clinical outcome in chronic myelogenous leukemia cancer patients.
  • This Example shows that the expression levels of the sternness signature genes is also associated with the progression of chronic myelogenous leukemia (CML) and thus, the sternness signature can be used to predict the progression of CML in a subject.
  • CML chronic myelogenous leukemia
  • Example 6 Inhibitory effect of ⁇ -secretase inhibitor on the expression of sternness signature genes in a variety of preclinical tumor models.
  • NOTCH signaling is deregulated in the majority of T-cell Acute Lymphoblastic Leukemias (T-ALL). Somatic, activating mutations in NOTCHl are present in >50% of patients with T-ALL (Weng et al., 2004 Science 306:269-71 ; Zhu et al., 2006 Clin. Cancer Res. 12:3043- 49).
  • GSIs Gamma Secretase Inhibitors
  • GSI sensitivity of a panel of TALL cells lines was measured in vitro and then correlated with the effect of the GSI on the sternness signature genes (Table 3). Sensitivity was determined as GIso values (growth inhibition) using an ATP-based viability assay after 7 days exposure to two GSIs: Compound S (MK-0752) (cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5- difluorophenyl) cyclohexanepropanoic acid sodium salt (WO2006123182; )); and Compound E ((S 5 S)- 2-[2-(3,5-Difluorophenyl)-acetylamino]-N-(l-methyl-2-oxo-5-phenyl-2,3-dihydro-lH- benzo[e][l,4]diazepin-3-yl)-propionamide) (purchased from Calbiochem, San D ⁇ ago, CA
  • the sternness signature score for each individual sample was determined by calculating unweighted average of log 10 ratio all 58 genes in the individual sample.
  • the greater the down- regulation of the sternness signature genes in T-ALL cell lines, as represented by the sternness score, the lower the IC 50 of ⁇ -secretase inhibitor for growth inhibition. This suggests that both the magnitude and duration of down-regulation of the sternness signature genes are significantly associated with the growth inhibitory effect of ⁇ -secretase inhibition in T-ALL cells in culture (correlation coefficient 0.68, P 3.3X10 's ).
  • mice used in this study contained the following genetic elements: Ink4a/Arf /' MMTV-rtTA, TetO-Her2 v659E , and TetO- Luciferase. These genetically engineered mice develop mammary tumors and provide a model of Her2-amplified breast cancer. Like human breast cancers., tumors derived from these transgenic mice show diversity at the level of DNA copy number, gene expression, histology and in their response to therapeutic agents (Kannan et al. s 2007 American Association for Cancer Research).
  • Tumors derived from the Her2-activated, Ink4a-deleted mice were grown as sub-cutaneous allografts in Balb/c nude mice and treated with MRK-003, a cyclic sulfamide which is potent and specific GSI (Lewis et al., 2007; Sparey et al., 2005 Bioorg. Med. Chem. Lett. 15:4212-16). Animals were treated with vehicle or a single dose of 75mg/kg or 300mg/kg of MRK-003 (p.o.). RNA from treated and control tumors was extracted from dissected tumors 6 hours post-dose and subject to expression profiling.
  • cytotoxic chemotherapeutics are sometime effective in de-bulking tumors by non-specifically blocking rapidly dividing tumor cells, it is postulated that a sub-population of tumors with stem cell-like characteristics may evade these therapeutic agents and seed tumor reinitiation.
  • the sternness signature gene set described here was a marker of this population of cells, then one would expect that a cytotxic agent would not inhibit the expression of the sternness signature genes.
  • the response of sternness genes to paclitaxel treatment was examined in a variety of cell lines.
  • Figure 7, Panel A presents data showing that there was no significant number of sternness signature genes down-regulated in either cell culture panels or in the A549 xenograft model. This result is in agreement with the drug resistance hypothesis. Interestingly, a majority of sternness signature genes were up-regulated by paclitaxel (12.5 mg/kg) in the A549 xenograft model 8 hours after paclitacel treatment (Figure 7, Panel B), implying different sensitivity to paclitaxel between tumors with less or greater sternness activity. S ⁇ mmary
  • Example 1 A set of 58 sternness signature genes from expression profiles in response to perturbations affecting the self-renewal and pluripotency features of ES cells was identified in Example 1.
  • the expression pattern of the sternness signature genes is associated with an increase of stem cell numbers reported in the same animal model.
  • Example 4 shows, consistent with the Goldie-Coldman hypothesis of tumor drug resistance, that the 58 sternness signature genes are highly expressed in patients with basal-Iike pathological phenotype, a subtype responding poorly to current standard chemotherapy or other therapeutic agents. The chance of overall survival and metastasis free survival are also significantly associated with the expression level of the 58 sternness genes in breast cancer patient datasets.
  • the sternness signature genes were derived from normal ES cells. Importantly, the sternness signature genes were selected to reflect the self-renewal and pluripotency characteristics of ES cells and show significant association with progression and clinical outcome of tumors (Examples 4 and 5). These data support the idea that at least a subpopulation of tumor cells may 'hijack' the molecular machinery underlying capabilities of self-renewal and pluripotency of ES cells. Therefore, the sternness signature genes identified herein may reveal some molecular features shared by both ES cells and tumors.
  • Sternness characteristics i.e., capacity for cellular self-renewal and pluripotency, could be orthogonal to other general features of cellular activities, such as proliferation, differentiation and survival.
  • Melton and colleagues (2007, J CHn Invest. 117:2553-61) have supported the idea that pancreatic beta-cells, fully committed and differentiated cells, possess self-renewal capabilities.
  • cell cycles of human ES cells are reported to be regulated by Dicer despite their sternness characteristics (Hatfield, et al., 2005, Nature. 435 :974-78.).
  • ⁇ -secretase inhibition is in agreement with the emerging role of the Notch pathway in regulating stem cell division and mediating critical communication between stem cells and their microenv ⁇ ronment (Androutsellis-Theotokis et al., 2006 Nature 442:823-26).
  • paclitaxel did not show any concordant effect on inhibition of the sternness signature genes across a variety of cell lines.
  • the distinct effects on the expression of sternness signature genes by a ⁇ -secretase inhibitor and paclitaxel suggests that ⁇ -secretase inhibition may target tumor cells that are resistant to current standard therapeutics. Patients with basal-like breast cancers may benefit from combining a ⁇ - secretase inhibitor with the standard of care chemotherapy.

Abstract

L’invention concerne, selon un aspect, des procédés, des biomarqueurs et des signatures d’expression destinés à évaluer le degré auquel un échantillon cellulaire présente des propriétés de cellules souches. Selon un autre aspect, l’invention concerne des procédés de comparaison d’agents qui modulent la fonction de gènes en fonction du degré auquel l’agent module l’expression génique des biomarqueurs de souchitude. Selon un autre aspect, l’invention concerne des procédés de prévision de la réponse d’un sujet atteint d’un cancer à un traitement par un agent, fondés sur des modifications des niveaux d’expression génique de biomarqueurs de souchitude. Selon un autre aspect, l’invention concerne des procédés de prévision de l’efficacité thérapeutique d’un agent thérapeutique anticancéreux.
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Citations (1)

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US20070099209A1 (en) * 2005-06-13 2007-05-03 The Regents Of The University Of Michigan Compositions and methods for treating and diagnosing cancer

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US20070099209A1 (en) * 2005-06-13 2007-05-03 The Regents Of The University Of Michigan Compositions and methods for treating and diagnosing cancer

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