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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
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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

• the present invention belongs to the fields of medicine and relates to the use of genomic analysis to evaluate and treat ovarian cancer.
• this invention relates to the measurement of patterns of gene expression to determine the presence of ovarian cancer in a patients tissues.
• Ovarian cancer is one of the most common types of cancer that affects women in the United States, with a lifetime risk of approximately 1/70. See Whittemore, Gynecol. Oncol., Vol. 55, No. 3, Part 2, pp. S15-S19 (1994). It is a rapidly fatal disease usually detected late, with still no good method of prevention. The greatest risk factor for ovarian cancer is a family history of the disease, suggesting the strong influence of genetics. See Schildkraut and Thompson, Am. J. Epidemiol., Vol. 128, No. 3, pp. 456-466 (1988). Other factors such as demographic, lifestyle and reproductive factors have also been shown to contribute to the risk of ovarian cancer.
• genes apparently expressed at high levels, or with the biggest change in expression, may not always be the most relevant; it is conceivable that a small disruption of the very tight regulation of genes may have dramatic consequences, even when the level of expression is low.
• this invention provides a method to determine if a patient is afflicted with ovarian cancer comprising: a) obtaining a sample from the said patient; b) determining the levels of gene expression of two or more of the genes listed in Table 9 in the sample from the patient; c) comparing the levels of gene expression of the two or more genes determined in (b) to the levels of the same genes listed in Table 1 ; d) determining the degree of similarity (DOS) between the levels of gene expression of the two or more genes determined in (c); and e) determining from the DOS between the level of gene expression of the two or more genes the probability that the sample shows evidence of the presence of ovarian cancer in the patient.
• this invention provides a method wherein the levels of gene expression are determined for a subset of the genes listed in Table 9 comprising genes
• the invention employs a sample comprising cells obtained from the patient. These may be cells removed from a solid tumor in the said patient or, in a preferred embodiment, the sample comprises blood cells and serum drawn from the said patient. In a most preferred embodiment, the sample comprises a body fluid drawn from the patient.
• this invention employs a method of determining the level of gene expression comprising measuring the levels of protein expression product in the sample from the patient. This may be done in a variety of ways including, but not limited to, detecting the presence and level of the protein expression products using a reagent which specifically binds with the proteins, wherein the reagent may be selected from the group consisting of an antibody, an antibody derivative and an antibody fragment.
• this invention provides a method wherein the levels of expression in the sample are assessed by measuring the levels in the sample of the transcribed polynucleotides of the two or more gene in Table 9.
• These transcribed polynucleotide may be mRNA or complementary DNA (cDNA).
• this method would further include the step of amplifying the transcribed polynucleotide.
• this invention includes a method of treating a subject afflicted with ovarian cancer, the method comprising providing to cells of the subject an antisense oligonuceotide complimentary to one or more of the genes whose expression is up-regulated in ovarian cancer as shown in Table 8.
• this invention provides a method of inhibiting ovarian cancer in a subject at risk for developing ovarian cancer, the method comprising inhibiting expression of one or more of the genes shown in Table 8 to be up-regulated in ovarian cancer.
• kits for use in determining treatment strategy for a patient with suspected ovarian cancer comprising: a) a number (for example, two or more) of antibodies able to recognize and bind to the polypeptide expression product of the two or more of the genes in Table 9; b) a container suitable for containing the said antibodies and a sample of body fluid from the said individual wherein the antibody can contact the polypeptide expressed by the two or more genes shown in Table 9 if they are present; c) means to detect the combination of the said antibodies with the polypeptides expressed by the two or more genes shown in Table 9; and d) instructions for use and interpretation of the kit results.
• this invention provides a kit for use in determining the presence or absence of ovarian cancer in a patient comprising: a) a number (for example, two or more) of polynucleotides able to recognize and bind to the mRNA expression product of the two or more genes shown in Table 9; b) a container suitable for containing the said polynucleotides and a sample of body fluid from the said individual wherein the said polynucleotide can contact the mRNA, if it is present; c) means to detect the levels of combination of the said polynucleotide with the mRNA from the two or more genes shown in Table 9; and d) instructions for use and interpretation of the kit results.
• a number for example, two or more
• FIG. 1(A) Re-Classification of Samples Using Increasing Number of Probe Sets.
• FIG. 1 (B). Plot of Errors as a Function of Number of Probe Sets for Determination of
• FIG. 2 Determination of a Threshold CC Value for Classification of Ovarian Status.
• FIG. 3 Correlation of Test and Validation Biopsy Profiles with Mean Normal Profile for Different Size Probe Sets.
• N or T represent Normal or Tumor status, respectively,
• r is the PCC value of the probe set profile of the corresponding biopsy sample with the mean
• FIG. 4 Correlation of Biopsy Profiles with Mean of AH Normal Profiles for Different Size Probe Sets. N or T represent Normal or Tumor status, respectively, "r" is the PCC value of the probe set profile of the corresponding biopsy sample with the mean profile of all Normal samples. Samples are ordered from highest CC to lowest.
• the present invention provides methods to determine whether or not a sample from a patient including, but not limited to, biopsy tissue or blood, serum or some other body fluid from a patient, contains evidence of the presence of ovarian cancer in the patient.
• This invention is based, in part, on the discovery of approximately 900 genes which are differentially expressed in tissue from ovarian cancer as compared to normal tissue.
• This methods of this invention comprise measuring the activities of the approximately 900 or fewer genes that are shown to be differently-expressed in ovarian cancer as compared to normal tissue.
• This DOS could be determined by any procedure that produces a result whose value is a known function of the DOS between the two groups of numbers, i.e., the measured gene expression values of the two or more genes in tissue from an individual whose ovarian cancer status is unknown and to be determined and the measured gene expression values for the same two or more genes from individuals whose tissue is known to contain ovarian cancer and from individuals whose tissue is known not to contain ovarian cancer.
• DOS shall mean the extent to which the pattern of gene expression values are alike or numerically similar, as measured by a comparison of the values of gene expression determined by direct or indirect methods.
• the DOS would be determined by a mathematical calculation resulting in a correlation coefficient (CC).
• CC correlation coefficient
• PCC Pearson Correlation Coefficient
• the value of the DOS (PCC), so calculated, can then be directly related to the probability that the tissue sample is from a patient who does or does not have ovarian cancer. That is to say, the higher the patients' DOS (CC or PCC) as compared to the gene expression values from a patient who does not have ovarian cancer or the higher the DOS (CC or PCC) as compared to the gene expression values from a patient who does have ovarian cancer then the greater the probability that the patient does not or does have ovarian cancer, respectively.
• the value of the DOS can be used to determine probabilities for the presence of ovarian cancer.
• PCC DOS
• this would work as shown in FIG. 3, using the 28 predictor probe set (as described below) if the gene expression profile correlates with the mean normal (No Ovarian Cancer) profile with a CC ⁇ 0.920 the tissue sample is 63 times more likely to contain ovarian cancer then if the CC >0.920 [odds ratio (OR) 63 with 95% confidence interval (Cl): 3.3-1194.7].
• the PCC can be set to produce optional sensitivity. That is, to make the smallest possible number of false negatives (Ovarian Cancer misclassified as No Ovarian Cancer).
• the threshold is determined by setting the PCC to >0.955.
• the threshold is determined by setting the PCC to >0.955.
• 100% of patients with a CC of >0.955 as compared to the No Ovarian Cancer group did not have ovarian cancer and 100% of the patients whose CC were ⁇ 0.870, as compared to the No Ovarian Cancer group, did have ovarian cancer.
• one of skill in the art can choose a PCC that will either maximize sensitivity or maximize specificity or produce any desired ratio of false positives or false negatives.
• One of skill in the art can easily adjust their choice of PCC to the clinical situation to provide maximum benefit and safety to the patient.
• Another aspect of the of the invention are methods to treat ovarian cancer. These methods consist of various efforts to suppress the excess gene expression of the genes that have been found to be up-regulated in ovarian cancer. These genes are shown in Table 8. Methods to decrease the excess expression of these gene would include, but not be limited to, use of antisense DNA, siRNA and methods to complex and deactivate the protein expression products of these over-expressed genes. Methods of Measurement
• the gene expression of a selected group of the 900 genes is determined by measuring mRNA levels from tissue samples as described below.
• the gene expression can be measured more indirectly by measuring polypeptide gene expression products in tissues including, but not limited to, tumor and blood tissue.
• gene expression is measured by identifying the presence or amount of one or more proteins encoded by one of the genes listed in Table 9.
• the present invention also provides systems for detecting two or more markers of interest, e.g., two or more markers from Table 2. For example, where it is determined that a finite set of particular markers provides relevant information, a detection system is provided that detects the finite set of markers. For example, as opposed to detecting all genes expressed in a tissue with a generic microarray, a defined microarray or other detection technology is employed to detect the plurality, e.g., 28, 42, etc., of markers that define a biological condition, e.g., the presence or absence of ovarian cancer, etc.
• the present invention is not limited by the method in which gene expression biomarkers are detected or measured.
• mRNA, cDNA or protein is detected in tissue samples, e.g., biopsy samples.
• mRNA, cDNA or protein is detected in bodily fluids, e.g., serum, plasma, urine or saliva.
• a preferred embodiment of the invention provides that the method of the invention is performed ex vivo.
• the present invention further provides kits for the detection of these relevant gene expression biomarkers.
• protein or the polypeptide expression product is detected. Protein expression may be detected by any suitable method. In some embodiments, proteins are detected by binding of an antibody specific for the protein. For example, in some embodiments, antibody binding is detected using a suitable technique including, but not limited to, radioimmunoassay, enzyme-linked immunosorbant assay (ELISA), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, e.g., using colloidal gold, enzyme or radioisotope labels, e.g., Western blots, precipitation reactions, agglutination assays, e.g., gel agglutination assays, hemagglutination assays, etc., complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays and proteomic assays, such as the use of
• antibody binding is detected by detecting a label on the primary antibody.
• the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
• the secondary antibody is labeled.
• an automated detection assay is utilized. Methods for the automation of immunoassays include, but are not limited to, those described in U.S. Patent Nos. 5,885,530; 4,981,785; 6,159,750; and 5,358,691, each of which is herein incorporated by reference.
• the analysis and presentation of results is also automated.
• software that generates a diagnosis and/or prognosis based on the presence or absence of a series of proteins corresponding to markers is utilized.
• the immunoassay described in U.S. Patent Nos. 5,599,677 and 5,672,480, each of which is herein incorporated by reference, is utilized.
• proteins are detected by immunohistochemistry.
• markers are detected at the level of cDNA or RNA.
• gene expression biomarkers shall mean any biologic marker which can indicate the rate or degree of gene expression of a specific gene including, but not limited to, mRNA, cDNA or the polypeptide expression product of the specific gene.
• gene expression biomarkers are detected using a PCR-based assay.
• reverse-transcriptase PCR RT-PCR
• RNA is enzymatically converted to cDNA using a reverse-transcriptase enzyme.
• the cDNA is then used as a template for a PCR reaction.
• PCR products can be detected by any suitable method including, but not limited to, gel electrophoresis and staining with a DNA-specific stain or hybridization to a labeled probe.
• the quantitative RT-PCR with standardized mixtures of competitive templates method described in U.S. Patent Nos. 5,639,606; 5,643, 765; and 5,876,978, each of which is herein incorporated by reference, is utilized.
• gene expression biomarkers are detected using a hybridization assay.
• a hybridization assay the presence or absence of a marker is determined based on the ability of the nucleic acid from the sample to hybridize to a complementary nucleic acid molecule, e.g., an oligonucleotide probe.
• a complementary nucleic acid molecule e.g., an oligonucleotide probe.
• a variety of hybridization assays are available.
• hybridization of a probe to the sequence of interest is detected directly by visualizing a bound probe, e.g., a Northern or Southern assay.
• a Northern or Southern assay See, e.g., Ausabel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1991).
• DNA Southern
• RNA Northern
• the DNA or RNA is then cleaved with a series of restriction enzymes that cleave infrequently in the genome and not near any of the markers being assayed.
• the DNA or RNA is then separated, e.g., on an agarose gel, and transferred to a membrane.
• a labeled probe or probes e.g., by incorporating a radionucleotide, is allowed to contact the membrane under low-, medium- or high-stringency conditions. Unbound probe is removed and the presence of binding is detected by visualizing the labeled probe.
• the DNA chip assay is a GeneChip (Affymetrix, Santa Clara, CA). See, e.g., U.S. Patent Nos. 6,045,996; 5,925,525; and 5,858,659, each of which is herein incorporated by reference.
• the GeneChip technology uses miniaturized, high-density arrays of oligonucleotide probes affixed to a "chip". Probe arrays are manufactured by Affymetrix's light-directed chemical synthesis process, which combines solid-phase chemical synthesis with photolithographic fabrication techniques employed in the semiconductor industry.
• the process constructs high-density arrays of oligonucleotides, with each probe in a predefined position in the array.
• Multiple probe arrays are synthesized simultaneously on a large glass wafer. The wafers are then diced, and individual probe arrays are packaged in injection-molded plastic cartridges, which protect them from the environment and serve as chambers for hybridization.
• the nucleic acid to be analyzed is isolated, amplified by PCR and labeled with a fluorescent reporter group.
• the labeled DNA is then incubated with the array using a fluidics station.
• the array is then inserted into the scanner, where patterns of hybridization are detected.
• the hybridization data are collected as light emitted from the fluorescent reporter groups already incorporated into the target, which is bound to the probe array. Probes that perfectly match the target generally produce stronger signals than those that have mismatches. Since the sequence and position of each probe on the array are known, by complementary, the identity of the target nucleic acid applied to the probe array can be determined.
• a DNA microchip containing electronically captured probes (Nanogen, San Diego, CA) is utilized. See, e.g., U.S. Patent Nos. 6,017,696; 6,068,818; and 6,051,380, each of which are herein incorporated by reference.
• Nanogen's technology enables the active movement and concentration of charged molecules to and from designated test sites on its semiconductor microchip.
• DNA capture probes unique to a given gene expression biomarkers are electronically placed at, or "addressed" to, specific sites on the microchip. Since nucleic acid molecules have a strong negative charge, they can be electronically moved to an area of positive charge.
• an array technology based upon the segregation of fluids on a flat surface (chip) by differences in surface tension (ProtoGene, Palo Alto, CA) is utilized. See, e.g., U.S. Patent Nos. 6,001,311; 5,985,551; and 5,474,796, each of which is herein incorporated by reference.
• Protogene's technology is based on the fact that fluids can be segregated on a flat surface by differences in surface tension that have been imparted by chemical coatings. Once so segregated, oligonucleotide probes are synthesized directly on the chip by ink-jet printing of reagents.
• a "bead array” is used for the detection of gene expression biomarkers (lllumina, San Diego, CA). See, e.g., PCT Publications WO 99/67641 and WO 00/39587, each of which is herein incorporated by reference, lllumina uses a BEAD ARRAY technology that combines fiber optic bundles and beads that self-assemble into an array. Each fiber optic bundle contains thousands to millions of individual fibers depending on the diameter of the bundle. The beads are coated with an oligonucleotide specific for the detection of a given marker. Batches of beads are combined to form a pool specific to the array. To perform an assay, the BEAD ARRAY is contacted with a prepared sample. Hybridization is detected using any suitable method.
• hybridization is detected by enzymatic cleavage of specific structures, e.g., INVADERTM assay, Third Wave Technologies. See, e.g., U.S. Patent Nos. 5,846,717, 6,090, 543; 6,001,567; 5,985,557; and 5,994,069, each of which is herein incorporated by reference.
• hybridization of a bound probe is detected using a TaqMan assay (PE Biosystems, Foster City, CA). See, e.g., U.S. Patent Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by reference. The assay is performed during a PCR reaction.
• the TaqMan assay exploits the 5'-3' exonuclease activity of DNA polymerases, such as AMPLITAQ DNA polymerase.
• a probe specific for a given marker, is included in the PCR reaction.
• the probe consists of an oligonucleotide with a 5'-reporter dye, e.g., a fluorescent dye and a 3'-quencher dye.
• a 5'-reporter dye e.g., a fluorescent dye and a 3'-quencher dye.
• the 5'-3' nucleolytic activity of the AMPLITAQ polymerase cleaves the probe between the reporter and the quencher dye.
• the separation of the reporter dye from the quencher dye results in an increase of fluorescence.
• the signal accumulates with each cycle of PCR and can be monitored with a fluorimeter.
• Additional detection assays that are produced and utilized using the systems and methods of the present invention include, but are not limited to, enzyme mismatch cleavage methods, e.g., Variagenics (see U.S. Patent Nos. 6,110,684; 5,958,692; and 5,851,770, herein incorporated by reference in their entireties); branched hybridization methods, e.g., Chiron (see U.S. Patent Nos. 5,849,481; 5,710,264; 5,124,246; and 5,624,802, herein incorporated by reference in their entireties); rolling circle replication (see, e.g., U.S. Patent Nos.
• mass spectroscopy is used to detect gene expression biomarkers.
• a MASSARRAYTM system (Sequenom, San Diego, CA) is used to detect gene expression biomarkers. See, e.g., U.S. Patent Nos. 6,043,031; 5,777,324; and 5,605,798, each of which is herein incorporated by reference.
• kits for the identification, characterization and quantitation of gene expression biomarkers contain antibodies specific for gene expression biomarkers, in addition to detection reagents and buffers.
• the kits contain reagents specific for the detection of nucleic acid, e.g., oligonucleotide probes or primers.
• the kits contain all of the components necessary to perform a detection assay, including all controls, directions for performing assays and any necessary software for analysis and presentation of results.
• the kits contain instructions including a statement of intended use as required by the Environmental Protection Agency or U.S. Food and Drug Administration (FDA) for the labeling of in vitro diagnostic assays and/or of pharmaceutical or food products.
• FDA Environmental Protection Agency
• the present invention is a method of screening a test compound for the ability to inhibit, retard, reverse or mimic the gene expression changes characteristic of ovarian cancer.
• a test mammal known to have ovarian cancer with a test compound and then analyze a representative tissue of the mammal for the level of expression of the genes or sequences which change in expression in response to ovarian cancer.
• the tissue is biopsy material from the tumor or, in a preferred embodiment, an easily obtainable tissue, such as blood or serum.
• Methods of increasing and decreasing expression would be known to one of skill in the art. Examples for supplementation of expression would include supplying the organism with additional copies of the gene. A preferred example for decreasing expression would include RNA antisense technologies or pharmaceutical intervention.
• the genes disclosed in Table 2 would be appropriate drug development targets. One would use the information presented in the present application for drug development by using currently existing, or by developing, pharmaceutical compounds that either mimic or inhibit the activity of the genes listed in Table 2, or the proteins encoded by these genes. Therefore, the gene expression biomarkers or genes disclosed herein represent targets for pharmaceutical development and gene therapy or RNA antisense therapy with the goal of suppressing the changes characteristic of ovarian cancer at the molecular level. These gene expression alterations may also play a role in understanding the various mechanisms that underlie ovarian cancer. Additionally, these genes represent biomarkers of ovarian cancer that can be used for diagnostic purposes.
• the present invention is not limited by the form of the expression profile.
• the expression profile is maintained in computer software.
• the expression profile is written material.
• the present invention is not limited by the number of markers provided or displayed in an expression profile.
• the expression profile may comprise two or more markers found in Table 2, indicating a biological status of a sample.
• the present invention further provides databases comprising expression information, e.g., expression profiles comprising one or more markers from Table 2 from one or more samples.
• the databases find use in data analysis including, but not limited to, comparison of markers to one or more public or private information databases, e.g., OMIM, GenBank, BLAST, Molecular Modeling Databases, Medline, genome databases, etc.
• an automated process is carried out to automatically associate information obtained from data obtained using the methods of the present invention to information in one or more of public or private databases. Associations find use, e.g., in making expression correlations to phenotypes, e.g., disease states.
• EXAMPLE 1 Preferred Methods To identify genes involved in the development and progression of ovarian tumors, we compared the gene expression profiles of a series of Normal and Tumor ovarian biopsies. Gene expression data for more than 12,000 genes were generated from each sample. Of the 900 probe sets that we observed to be most differentially-expressed between the Normal and cancerous ovarian biopsies, 98% were down-regulated in the Tumor biopsies. Using 8 Normal and 10 Tumor samples, we identified a minimum number of probe sets (28) that could be used to classify biopsies as Normal or Tumor. This finding was validated on a second set of biopsies (4 Normal and 14 Tumor) previously profiled by another laboratory. A mean Normal ovarian profile was established that could be used as a reference to compare other ovarian biopsies. The identification of the most differentially-expressed genes between
• Normal and Tumor ovarian biopsies may provide new insight into the molecular mechanisms of ovarian tumor development and progression.
• Some of the genes identified in this study are known to be involved in ovarian cancer, but a large proportion represents novel candidates for drug targets and molecular biomarkers to diagnose or monitor disease and treatment.
• Flash-frozen ovarian biopsies were obtained from Asterand (Detroit, Ml), and consisted of 10 Tumor samples and 10 adjacent Normal tissues. Total RNA was also purchased for 4 additional samples from Ambion (Austin, TX) and Stratagene (La Jolla, CA).
• tumors analyzed were malignant surface epithelial serous tumors, e.g., papillary cystcarcinoma, papillary cystadenocarcinoma or papillary cystcarcinoma; others included a mucinous cyst carcinoma, an endometrioid adenocarcinoma and a mature teratoma.
• Mote Paired samples (Normal and Tumor adjacent tissue) obtained from the same patient are boxed together. Stages of ovarian cancers are indicated using the FIGO staging system.
• Genome U95Av2 http://www.affvmetrix.com/products/arravs/specific/hau95.affx).
• a "false Neg” is defined as a Tumor incorrectly classified as a Normal ovary tissue
• a "false Pos” is defined as a Normal tissue incorrectly classified as a Tumor.
• the OR indicates that an ovarian biopsy of the test set is nearly 63 times more likely to be from an ovarian tumor if its expression profile of the 28 predictor probe sets correlates with the mean Normal profile with a CC ⁇ 0.920 (see Table 4). In our test set, 100% of profiles with a CC >0.955 correspond to Normal biopsies and 100% of profiles with a CC ⁇ 0.870 correspond to Tumor biopsies (see Fig. 3). Validation of the mean Normal profile
• Fig. 3 summarizes the classification of all ovarian biopsies based on the correlation of 28 probe sets. Remarkably, the profiles of the Normal and Tumor samples of the validation set were clearly separated from each other (see Fig. 3). As in the test set, 100% of profiles with a CC ⁇ 0.870 correspond to Tumor biopsies.
• Probe sets were ranked from highest absolute PCC to lowest, first using the 18 samples from the test set, and then with all 36 samples from both the test set and the validation set. From the 900 probe sets selected, 694 and 473 had an absolute CC higher than 0.5 with the 18 and 36 samples, respectively; 412 probe sets had a coefficient higher than 0.5 in both cases. Interestingly, from the 28 probe sets originally selected for the biopsy classification, 19 ranked in the top 100; the other 9 probe sets had correlation values ranging from 0.359-0.703.
• Claudin 4 a component of tight junctions, has been shown to be up-regulated in ovarian tumors together with another member of this family of transmembrane receptors, Claudin 3.
• Claudin 3 Another member of this family of transmembrane receptors, Claudin 3.
• Costa and colleagues have reported that levels of topoisomerase II alpha correlate with poor prognosis of ovarian surface epithelial neoplasms.
• Kallikrein 8 has been detected by immunohistochemistry in carcinoma but not Normal ovarian tissue and was suggested as a prognostic marker of ovarian cancer. See Underwood et al., Cancer Res., Vol. 59, No. 17, pp.
• TGF ⁇ R3 transforming growth factor beta receptor III
• PDGFRL platelet-derived growth factor receptor-like gene
• ST13 tumorigenicity gene
• RECK reversion-inducing- cysteine-rich protein with kazal motif
• PEG3 paternally expressed 3
• biopsies and, in particular, Tumor biopsies have a substantial level of heterogeneity: tumor type, grade, percentage of tumor cells, presence of connective and fat tissues, etc.
• We studied different types of ovarian tumors of various grades see Table 1) to search for genes involved in common pathways of tumor development and progression, rather than genes involved more specifically in certain types of tumors as previously reported. See Ono et al. (2000), supra; and Welsh et al. (2001), supra.
• RNA abundances and activities are currently fall within three classes: ribozymes, antisense species and RNA aptamers. See Good et al., Gene Ther., Vol. 4, No. 1 , pp. 45-54 (1997). Controllable application or exposure of a cell to these entities permits controllable perturbation of RNA abundances. Ribozymes
• Ribozymes are RNAs which are capable of catalyzing RNA cleavage reactions. See Cech, Science, Vol. 236, pp. 1532-1539 (1987); PCT International Publication WO 90/11364 (1990); Sarver et al., Science, Vol. 247, pp. 1222-1225 (1990). "Hairpin” and "hammerhead” RNA ribozymes can be designed to specifically cleave a particular target mRNA. Rules have been established for the design of short RNA molecules with ribozyme activity, which are capable of cleaving other RNA molecules in a highly sequence specific way and can be targeted to virtually all kinds of RNA. See Haseloff et al., Nature, Vol.
• Ribozyme methods involve exposing a cell to, inducing expression in a cell, etc. of such small RNA ribozyme molecules. See Grassi and Marini, Annals of Med., Vol. 28, No. 6, pp.499-510 (1996); and Gibson, Cancer Meta. Rev., Vol. 15, pp. 287-299 (1996).
• Ribozymes can be routinely expressed in vivo in sufficient number to be catalytically effective in cleaving mRNA, and thereby modifying mRNA abundances in a cell. See Cotton et al., EMBO J., Vol. 8, pp. 3861-3866 (1989).
• a ribozyme coding DNA sequence designed according to the previous rules and synthesized, e.g., by standard phosphoramidite chemistry, can be ligated into a restriction enzyme site in the anticodon stem and loop of a gene encoding a tRNA, which can then be transformed into and expressed in a cell of interest by methods routine in the art.
• an inducible promoter e.g., a glucocorticoid or a tetracycline esponse element
• an inducible promoter e.g., a glucocorticoid or a tetracycline esponse element
• a highly and constituently active promoter can be used.
• tDNA genes i.e., genes encoding tRNAs, are useful in this application because of their small size, high rate of transcription and ubiquitous expression in different kinds of tissues.
• ribozymes can be routinely designed to cleave virtually any mRNA sequence, and a cell can be routinely transformed with DNA coding for such ribozyme sequences such that a controllable and catalytically effective amount of the ribozyme is expressed. Accordingly, the abundance of virtually any RNA species in a cell can be modified or perturbed.
• Antisense molecules can be routinely designed to cleave virtually any mRNA sequence, and a cell can be routinely transformed with DNA coding for such ribozyme sequences such that a controllable and catalytically effective amount of the ribozyme is expressed. Accordingly, the abundance of virtually any RNA species in a cell can be modified or perturbed.
• activity of a target RNA (preferably mRNA) species can be controllably inhibited by the controllable application of antisense nucleic acids.
• Application at high levels results in a saturating inhibition.
• An "antisense" nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a sequence-specific, e.g., non-poly A, portion of the target RNA, e.g., its translation initiation region, by virtue of some sequence complementary to a coding and/or non-coding region.
• the antisense nucleic acids of the invention can be oligonucleotides that are double- stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered in a controllable manner to a cell or which can be produced intracellularly by transcription of exogenous, introduced sequences in controllable quantities sufficient to perturb translation of the target RNA.
• antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides, ranging from 6 oligonucleotides to about 200 oligonucleotides.
• the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides or at least 200 nucleotides.
• the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded.
• the oligonucleotide can be modified at the base moiety, sugar moiety or phosphate backbone.
• the oligonucleotide may include other appending groups, such as peptides, or agents facilitating transport across the cell membrane [see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA, Vol. 84, pp. 648-652 (1987); and PCT Publication No. WO 88/09810 (1988)], hybridization-triggered cleavage agents [see, e.g., Krol et al., BioTechniques, Vol. 6, pp. 958- 976 (1988)] or intercalating agents [see, e.g., Zon, Pharm. Res., Vol. 5, No. 9, pp. 539-549 (1988)].
• an antisense oligonucleotide is provided, preferably as single-stranded DNA.
• the oligonucleotide may be modified at any position on its structure with constituents generally known in the art.
• the antisense oligonucleotides may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, ⁇ -D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, ⁇ -D-
• the oligonucleotide comprises at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose and hexose.
• the oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester and a formacetal or analog thereof.
• the oligonucleotide is a 2-a-anomeric oligonucleotide.
• oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual B-units, the strands run parallel to each other. See Gautier et al., Nucl. Acids Res., Vol. 15, pp. 6625-6641 (1987).
• the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
• the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of a target RNA species.
• absolute complementary although preferred, is not required.
• the ability to hybridize will depend on both the degree of complementary and the length of the antisense nucleic acid.
• Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer, such as are commercially-available from Biosearch, Applied Biosystems, etc.
• phosphorothioate oligonucleotides may be synthesized by the method of Stein et al., Nucl. Acids Res., Vol. 16, p. 3209 (1988), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports, etc. See Sarin et al., Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 7448-7451 (1988).
• the oligonucleotide is a 2'-0-methylribonucleotide [see Inoue et al., Nucl. Acids Res., Vol. 15, pp. 6131-6148 (1987)] or a chimeric RNA-DNA analog [see Inoue et al., FEBS Lett., Vol. 215, pp. 327-330 (1987)].
• the synthesized antisense oligonucleotides can then be administered to a cell in a controlled or saturating manner.
• the antisense oligonucleotides can be placed in the growth environment of the cell at controlled levels where they may be taken up by the cell.
• the uptake of the antisense oligonucleotides can be assisted by use of methods well- known in the art.
• the antisense nucleic acids of the invention are controllably expressed intracellularly by transcription from an exogenous sequence. If the expression is controlled to be at a high level, a saturating perturbation or modification results.
• a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
• RNA antisense nucleic acid
• Such a vector would contain a sequence encoding the antisense nucleic acid.
• Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
• Vectors can be constructed by recombinant DNA technology methods standard in the art.
• Vectors can be plasmid, viral or others known in the art, used for replication and expression in mammalian cells.
• Expression of the sequences encoding the antisense RNAs can be by any promoter known in the art to act in a cell of interest.
• promoters can be inducible or constitutive.
• promoters are controllable or inducible by the administration of an exogenous moiety in order to achieve controlled expression of the antisense oligonucleotide.
• controllable promoters include the Tet promoter.
• promoters for mammalian cells include, but are not limited to, the SV40 early promoter region [see Bernoist and Chambon, Nature, Vol. 290, pp. 304-310 (1981)], the promoter contained in the 3' long terminal repeat of Rous sarcoma virus [see Yamamoto et al., Ceil, Vol. 22, pp. 787-797 (1980)], the herpes thymidine kinase promoter [see Wagner et al., Proc. Natl. Acad. Sci. USA, Vol. 78, pp. 1441-1445 (1981)], the regulatory sequences of the metallothionein gene, etc. [see Brinster et al., Nature, Vol. 296, pp. 39-42 (1982)].
• antisense nucleic acids can be routinely designed to target virtually any mRNA sequence, and a cell can be routinely transformed with or exposed to nucleic acids coding for such antisense sequences such that an effective and controllable or saturating amount of the antisense nucleic acid is expressed. Accordingly the translation of virtually any RNA species in a cell can be modified or perturbed.
• RNA Aptamers
• RNA aptamers can be introduced into or expressed in a cell.
• RNA aptamers are specific RNA ligands for proteins, such as for Tat and Rev RNA [see Good et al. (1997), supra] that can specifically inhibit their translation.
• Methods of modifying protein abundances include, inter alia, those altering protein degradation rates and those using antibodies, which bind to proteins affecting abundances of activities of native target protein species. Increasing (or decreasing) the degradation rates of a protein species decreases (or increases) the abundance of that species. Methods for increasing the degradation rate of a target protein in response to elevated temperature and/or exposure to a particular drug, which are known in the art, can be employed in this invention.
• one such method employs a heat-inducible or drug-inducible /V-terminal degron, which is an ⁇ /-terminal protein fragment that exposes a degradation signal promoting rapid protein degradation at a higher temperature, e.g., 37°C, and which is hidden to prevent rapid degradation at a lower temperature, e.g., 23°C.
• a heat-inducible or drug-inducible /V-terminal degron which is an ⁇ /-terminal protein fragment that exposes a degradation signal promoting rapid protein degradation at a higher temperature, e.g., 37°C, and which is hidden to prevent rapid degradation at a lower temperature, e.g., 23°C.
• a degron is Arg-DHFR ts , a variant of murine dihydrofolate reductase in which the /V-terminal Val is replaced by Arg and the Pro at position 66 is replaced with Leu.
• a gene for a target protein, P is replaced by standard gene targeting methods known in the art [see Lodish et al., Molecular Biology of the Cell, W.H. Freeman and Co., NY, especially Chapter 8 (1995)] with a gene coding for the fusion protein Ub-Arg-DHFR te -P ("Ub” stands for ubiquitin).
• Ub stands for ubiquitin
• the /V-terminal ubiquitin is rapidly cleaved after translation exposing the /V-terminal degron. At lower temperatures, lysines internal to Arg-DHFR ts are not exposed, ubiquitination of the fusion protein does not occur, degradation is slow and active target protein levels are high.
• Target protein activities can also be decreased by (neutralizing) antibodies.
• protein abundances/activities can be modified or perturbed in a controlled or saturating manner.
• antibodies to suitable epitopes on protein surfaces may decrease the abundance, and thereby indirectly decrease the activity, of the wild-type active form of a target protein by aggregating active forms into complexes with less or minimal activity as compared to the wild-type unaggregated wild-type form.
• antibodies may directly decrease protein activity by, e.g., interacting directly with active sites or by blocking access of substrates to active sites.
• (activating) antibodies may also interact with proteins and their active sites to increase resulting activity.
• antibodies of the various types to be described
• the effects of the antibodies can be assayed and suitable antibodies selected that raise or lower the target protein species concentration and/or activity.
• Such assays involve introducing antibodies into a cell (see below) and assaying the concentration of the wild-type amount or activities of the target protein by standard means, such as immunoassays, known in the art.
• the net activity of the wild-type form can be assayed by assay means appropriate to the known activity of the target protein.
• Antibodies can be introduced into cells in numerous fashions, including, e.g., microinjection of antibodies into a cell [see Morgan et al., Immunol.
• recombinant antibodies can be engineering and ectopically expressed in a wide variety of non-lymphoid cell types to bind to target proteins, as well as to block target protein activities. See Biocca et al., Trends Cell Biol., Vol. 5, pp. 248-252 (1995). Expression of the antibody is preferably under control of a controllable promoter, such as the Tet promoter, or a constitutively active promoter (for production of saturating perturbations).
• a controllable promoter such as the Tet promoter, or a constitutively active promoter (for production of saturating perturbations).
• a first step is the selection of a particular monoclonal antibody with appropriate specificity to the target protein (see below). Then sequences encoding the variable regions of the selected antibody can be cloned into various engineered antibody formats, including, e.g., whole antibody, Fab fragments, Fv fragments, single-chain Fv (ScFv) fragments (V H and V L regions united by a peptide linker), diabodies (two associated ScFv fragments with different specificities) and so forth. See Hayden et al., Curr. Opin. Immunol., Vol. 9, pp. 210-212 (1997).
• Intracellularly-expressed antibodies of the various formats can be targeted into cellular compartments, e.g., the cytoplasm, the nucleus, the mitochondria, etc., by expressing them as fusions with the various known intracellular leader sequences. See Bradbury et al., Antibody Engineerinq, Borrebaeck, Editor, Vol. 2, pp. 295-361, IRL Press (1995).
• the ScFv format appears to be particularly suitable for cytoplasmic targeting.
• Antibody types include, but are not limited to, polyclonal, monoclonal, chimeric, single-chain, Fab fragments and an Fab expression library.
• Various procedures known in the art may be used for the production of polyclonal antibodies to a target protein.
• various host animals can be immunized by injection with the target protein, such host animals include, but are not limited to, rabbits, mice, rats, etc.
• adjuvants can be used to increase the immunological response, depending on the host species and include, but are not limited to, Freund's (complete and incomplete); mineral gels, such as aluminum hydroxide; surface active substances, such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol; and potentially useful human adjuvants, such as Bacillus Calmette-Guerin (BCG) and corynebacterium parvum.
• BCG Bacillus Calmette-Guerin
• any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. Such techniques include, but are not restricted to, the hybridoma technique originally developed by Kohler and Milstein, Nature, Vol.
• monoclonal antibodies can be produced in germ-free animals utilizing recent technology. See PCT/US90/02545.
• human antibodies may be used and can be obtained by using human hybridomas [see Cote et al., Proc. Natl.
• monoclonal antibodies can be alternatively selected from large antibody libraries using the techniques of phage display. See Marks et al., J. Biol. Chem., Vol. 267, pp. 16007-16010 (1992). Using this technique, libraries of up to 10 12 different antibodies have been expressed on the surface of fd filamentous phage, creating a "single pot" in vitro immune system of antibodies available for the selection of monoclonal antibodies. See Griffiths et al., EMBO J., Vol. 13, pp. 3245-3260 (1994).
• Selection of antibodies from such libraries can be done by techniques known in the art, including contacting the phage to immobilized target protein, selecting and cloning phage bound to the target and subcloning the sequences encoding the antibody variable regions into an appropriate vector expressing a desired antibody format.
• Antibody fragments that contain the idiotypes of the target protein can be generated by techniques known in the art.
• such fragments include, but are not limited to, the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments that can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, the Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent and Fv fragments.
• screening for the desired antibody can be accomplished by techniques known in the art, e.g., ELISA.
• ELISA ELISA-Linked Immunosorbent Assays
• Methods of directly modifying protein activities include, inter alia, dominant negative mutations, specific drugs or chemical moieties and also the use of antibodies, as previously discussed.
• Dominant negative mutations are mutations to endogenous genes or mutant exogenous genes that when expressed in a cell disrupt the activity of a targeted protein species. Depending on the structure and activity of the targeted protein, general rules exist that guide the selection of an appropriate strategy for constructing dominant negative mutations that disrupt activity of that target. See Hershkowitz, Nature, Vol. 329, pp. 219-222 (1987). In the case of active monomeric forms, over expression of an inactive form can cause competition for natural substrates or ligands sufficient to significantly reduce net activity of the target protein.
• over expression can be achieved by, e.g., associating a promoter, preferably a controllable or inducible promoter, or also a constitutively expressed promoter, of increased activity with the mutant gene.
• a promoter preferably a controllable or inducible promoter, or also a constitutively expressed promoter
• changes to active site residues can be made so that a virtually irreversible association occurs with the target ligand.
• Such can be achieved with certain tyrosine kinases by careful replacement of active site serine residues. See Perlmutter et al., Curr. Opin. Immunol., Vol. 8, pp. 285-290 (1996).
• several strategies can guide selection of a dominant negative mutant.
• Multimeric activity can be decreased in a controlled or saturating manner by expression of genes coding exogenous protein fragments that bind to multimeric association domains and prevent multimer formation.
• controllable or saturating over-expression of an inactive protein unit of a particular type can tie up wild-type active units in inactive multimers, and thereby decrease multimeric activity.
• the DNA binding domain can be deleted from the DNA binding unit, or the activation domain deleted from the activation unit.
• the DNA binding domain unit can be expressed without the domain causing association with the activation unit.
• DNA binding sites are tied up without any possible activation of expression.
• a particular type of unit normally undergoes a conformational change during activity
• expression of a rigid unit can inactivate resultant complexes.
• proteins involved in cellular mechanisms such as cellular motility, the mitotic process, cellular architecture and so forth, are typically composed of associations of many subunits of a few types. These structures are often highly sensitive to disruption by inclusion of a few monomeric units with structural defects. Such mutant monomers disrupt the relevant protein activities and can be expressed in a cell in a controlled or saturating manner.
• mutant target proteins that are sensitive to temperature (or other exogenous factors) can be found by mutagenesis and screening procedures that are well-known in the art.
• activities of certain target proteins can be modified or perturbed in a controlled or a saturating manner by exposure to exogenous drugs or ligands. Since the methods of this invention are often applied to testing or confirming the usefulness of various drugs to treat cancer, drug exposure is an important method of modifying/perturbing cellular constituents, both mRNAs and expressed proteins.
• input cellular constituents are perturbed either by drug exposure or genetic manipulation, such as gene deletion or knockout; and system responses are measured by gene expression technologies, such as hybridization to gene transcript arrays (described in the following).
• a drug that interacts with only one target protein in the cell and alters the activity of only that one target protein, either increasing or decreasing the activity.
• Graded exposure of a cell to varying amounts of that drug thereby causes graded perturbations of network models having that target protein as an input. Saturating exposure causes saturating modification/perturbation.
• Cyclosporin A is a very specific regulator of the calcineurin protein, acting via a complex with cyclophilin.
• a titration series of Cyclosporin A therefore can be used to generate any desired amount of inhibition of the calcineurin protein.
• saturating exposure to Cyclosporin A will maximally inhibit the calcineurin protein.
• the experimental methods of this invention depend on measurements of cellular constituents.
• the cellular constituents measured can be from any aspect of the biological state of a cell. They can be from the transcriptional state, in which RNA abundances are measured, the translation state, in which protein abundances are measured, the activity state, in which protein activities are measured.
• the cellular characteristics can also be from mixed aspects, e.g., in which the activities of one or more proteins are measured along with the RNA abundances (gene expressions) of other cellular constituents.
• This section describes exemplary methods for measuring the cellular constituents in drug or pathway responses. This invention is adaptable to other methods of such measurement.
• the transcriptional state of the other cellular constituents are measured.
• the transcriptional state can be measured by techniques of hybridization to arrays of nucleic acid or nucleic acid mimic probes, described in the next subsection, or by other gene expression technologies, described in the subsequent subsection.
• the result is data including values representing mRNA abundance and/or ratios, which usually reflect DNA expression ratios (in the absence of differences in RNA degradation rates).
• aspects of the biological state other than the transcriptional state such as the translational state, the activity state or mixed aspects can be measured.
• measurements of the cellular constituents should be made in a manner that is relatively independent of when the measurement are made.
• measurement of the transcriptional state is made by hybridization to transcript arrays, which are described in this subsection. Certain other methods of transcriptional state measurement are described later in this subsection.
• transcript arrays also called herein “microarrays”.
• Transcript arrays can be employed for analyzing the transcriptional state in a cell, and especially for measuring the transcriptional states of cancer cells.
• transcript arrays are produced by hybridizing detectably-labeled polynucleotides representing the mRNA transcripts present in a cell, e.g., fluorescently- labeled cDNA synthesized from total cell mRNA, to a microarray.
• a microarray is a surface with an ordered array of binding, e.g., hybridization, sites for products of many of the genes in the genome of a cell or organism, preferably most or almost all of the genes.
• Microarrays can be made in a number of ways, of which several are described below. However produced, microarrays share certain characteristics. The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other.
• the microarrays are small, usually smaller than 5 cm 2 and they are made from materials that are stable under binding, e.g. nucleic acid hybridization, conditions.
• a given binding site or unique set of binding sites in the microarray will specifically bind the product of a single gene in the cell.
• site physical binding site
• positionally-addressable arrays containing affixed nucleic acids of known sequence at each location are used.
• cDNA complementary to the total cellular mRNA is hybridized to a microarray
• the site on the array corresponding to a gene i.e., capable of specifically binding the product of the gene, that is not transcribed in the cell will have little or no signal, e.g., fluorescent signal, and a gene for which the encoded mRNA is prevalent will have a relatively strong signal.
• Microarrays are known in the art and consist of a surface to which probes that correspond in sequence to gene products, e.g., cDNAs, mRNAs, cRNAs, polypeptides and fragments thereof, can be specifically hybridized or bound at a known position.
• the microarray is an array, i.e., a matrix, in which each position represents a discrete binding site for a product encoded by a gene, e.g., a protein or RNA, and in which binding sites are present for products of most or almost all of the genes in the organism's genome.
• the "binding site”, hereinafter “site”, is a nucleic acid or nucleic acid analogue to which a particular cognate cDNA can specifically hybridize.
• the nucleic acid or analogue of the binding site can be, e.g., a synthetic oligomer, a full-length cDNA, a less than full-length cDNA or a gene fragment.
• the microarray contains binding sites for products of all or almost all genes in the target organism's genome, such comprehensiveness is not necessarily required.
• the microarray will have binding sites corresponding to at least about 50% of the genes in the genome, often at least about 75%, more often at least about 85%, even more often more than about 90%, and most often at least about 99%.
• the microarray has binding sites for genes relevant to testing and confirming a biological network model of interest.
• a "gene” is identified as an open reading frame (ORF) of preferably at least 50, 75 or 99 amino acids from which a mRNA is transcribed in the organism, e.g., if a single cell, or in some cell in a multicellular organism.
• ORF open reading frame
• the number of genes in a genome can be estimated from the number of mRNAs expressed by the organism, or by extrapolation from a well-characterized portion of the genome.
• the number of ORFs can be determined and mRNA coding regions identified by analysis of the DNA sequence.
• Saccharomyces cerevisiae genome has been completely sequenced and is reported to have approximately 6,275 ORFs longer than 99 amino acids. Analysis of these ORFs indicates that there are 5,885 ORFs that are likely to specify protein products. See Goffeau et al., Science, Vol. 274, pp. 546-567 (1996), which is incorporated by reference in its entirety for all purposes.
• the human genome is estimated to contain approximately 10 5 genes.
• the "binding site" to which a particular cognate cDNA specifically hybridizes is usually a nucleic acid or nucleic acid analogue attached at that binding site.
• the binding sites of the microarray are DNA polynucleotides corresponding to at least a portion of each gene in an organism's genome. These DNAs can be obtained by, e.g., PCR amplification of gene segments from genomic DNA, cDNA, e.g., by RT-PCR, or cloned sequences.
• PCR primers are chosen, based on the known sequence of the genes or cDNA, that result in amplification of unique fragments, i.e., fragments that do not share more than 10 bases of contiguous identical sequence with any other fragment on the microarray.
• Computer programs are useful in the design of primers with the required specificity and optimal amplification properties. See, e.g., Oligo pi version 5.0, National Biosciences.
• Oligo pi version 5.0 National Biosciences.
• it will sometimes be desirable to amplify segments near the 3' end of the gene so that when oligo-dT primed cDNA probes are hybridized to the microarray, less-than-full length probes will bind efficiently.
• each gene fragment on the microarray will be between about 50 bp and about 2000 bp, more typically between about 100 bp and about 1000 bp, and usually between about 300 bp and about 800 bp in length.
• PCR methods are well-known and are described, e.g., in Innis et al., eds., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, CA (1990), which is incorporated by reference in its entirety for all purposes. It will be apparent that computer-controlled robotic systems are useful for isolating and amplifying nucleic acids.
• nucleic acid for the microarray is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using ⁇ /-phosphonate or phosphoramidite chemistries. See Froehler et al., Nucleic Acid Res., Vol. 14, pp. 5399-5407 (1986); and McBride et al., Tetrahedron Lett., Vol. 24, pp. 245-248 (1983). Synthetic sequences are between about 15 bases and about 500 bases in length, more typically between about 20 bases and about 50 bases.
• synthetic nucleic acids include non-natural bases, e.g., inosine.
• nucleic acid analogues may be used as binding sites for hybridization.
• An example of a suitable nucleic acid analogue is peptide nucleic acid. See, e.g., Egholm et al., Nature, Vol. 365, pp. 566-568 (1993); and also U.S. Patent No. 5,539,083.
• the binding (hybridization) sites are made from plasmid or phage clones of genes, cDNAs, e.g., expressed sequence tags, or inserts therefrom. See Nguyen et al., Genomics, Vol. 29, pp. 207-209 (1995).
• the polynucleotide of the binding sites is RNA. Attaching Nucleic Acids to the Solid Surface
• the nucleic acid or analogue are attached to a solid support, which may be made from glass, plastic, e.g., polypropylene and nylon, polyacrylamide, nitrocellulose or other materials.
• a preferred method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al., Science, Vol. 270, pp. 467-470 (1995). This method is especially useful for preparing microarrays of cDNA. See, also, DeRisi et al., Nat. Genet, Vol. 14, pp. 457-460 (1996); Shalon et al., Genome Res., Vol. 6, pp.
• a second preferred method for making microarrays is by making high-density oligonucleotide arrays. Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ [see Fodor et al., Science, Vol. 251 , pp. 767-773 (1991); Pease et al., Proc. Natl. Acad. Sci.
• oligonucleotides e.g., 20 mers, of known sequence are synthesized directly on a surface such as a derivatized glass slide. Usually, the array produced is redundant, with several oligonucleotide molecules per RNA. Oligonucleotide probes can be chosen to detect alternatively spliced mRNAs.
• microarrays may also be used. See Maskos and Southern, Nucleic Acids Res., Vol. 20, pp. 1679-1684 (1992).
• any type of array e.g., dot blots on a nylon hybridization membrane [see Sambrook et al., Molecular Cloning-A Laboratory Manual, 2 nd Edition, Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), which is incorporated in its entirety for all purposes], could be used, although, as will be recognized by those of skill in the art, very small arrays will be preferred because hybridization volumes will be smaller.
• RNA is extracted from cells of the various types of interest in this invention using guanidinium thiocyanate lysis followed by CsCI centrifugation. See Chirgwin et al., Biochemistry, Vol. 18, pp. 5294-5299 (1979).
• Poly(A) + RNA is selected by selection with oligo-dT cellulose. See Sambrook et al. (1989), supra.
• Cells of interest include wild-type cells, drug-exposed wild-type cells, cells with modified/perturbed cellular constituent(s), and drug-exposed cells with modified/perturbed cellular constituent(s).
• Labeled cDNA is prepared from mRNA by oligo dT-primed or random-primed reverse transcription, both of which are well-known in the art. See, e.g., Klug and Berger, Methods Enzymol., Vol. 52, pp. 316-325 (1987). Reverse transcription may be carried out in the presence of a dNTP conjugated to a detectable label, most preferably a fluorescently- labeled dNTP. Alternatively, isolated mRNA can be converted to labeled antisense RNA synthesized by in vitro transcription of double-stranded cDNA in the presence of labeled dNTPs. See Lockhart et al.
• the cDNA or RNA probe can be synthesized in the absence of detectable label and may be labeled subsequently, e.g., by incorporating biotinylated dNTPs or rNTP, or some similar means, e.g., photo-cross-linking a psoralen derivative of biotin to RNAs, followed by addition of labeled streptavidin, e.g., phycoerythrin- conjugated streptavidin or the equivalent.
• biotinylated dNTPs or rNTP or some similar means, e.g., photo-cross-linking a psoralen derivative of biotin to RNAs, followed by addition of labeled streptavidin, e.g., phycoerythrin- conjugated streptavidin or the equivalent.
• fluorophores include fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others. See, e.g., Kricka, Nonisotopic DNA Probe Techniques, Academic Press, San Diego, CA (1992). It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished.
• a label other than a fluorescent label is used.
• a radioactive label or a pair of radioactive labels with distinct emission spectra, can be used. See Zhao et al., Gene, Vol. 156, p. 207 (1995); and Pietu et al., Genome Res., Vol. 6, p. 492 (1996).
• use of radioisotopes is a less-preferred embodiment.
• labeled cDNA is synthesized by incubating a mixture containing 0.5 mM dGTP, dATP and dCTP plus 0.1 mM dTTP plus fluorescent deoxyribonucleotides, e.g., 0.1 mM Rhodamine 110 UTP (Perken Elmer Cetus) or 0.1 mM Cy3 dUTP (Amersham), with reverse transcriptase, e.g., SuperScript.TM. II, LTI Inc., at 42°C for 60 minutes.
• nucleic acid hybridization and wash conditions are chosen so that the probe "specifically binds" or “specifically hybridizes” to a specific array site, i.e., the probe hybridizes, duplexes or binds to a sequence array site with a complementary nucleic acid sequence but does not hybridize to a site with a non-complementary nucleic acid sequence.
• one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is ⁇ 25 bases, there are no mismatches using standard base-pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch.
• the polynucleotides are perfectly complementary (no mismatches). It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls. See, e.g., Shalon et al. (1996), supra; and Chee et al., supra.
• Optimal hybridization conditions will depend on the length, e.g., oligomer vs. polynucleotide >200 bases; and type, e.g., RNA, DNA and PNA, of labeled probe and immobilized polynucleotide or oligonucleotide.
• General parameters for specific, i.e., stringent, hybridization conditions for nucleic acids are described in Sambrook et al. (1996), supra; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-lnterscience, NY (1987), which is incorporated in its entirety for all purposes.
• the fluorescence emissions at each site of a transcript array can be, preferably, detected by scanning confocal laser microscopy.
• a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used.
• a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously. See Shalon el al. (1996), supra, which is incorporated by reference in its entirety for all purposes.
• the arrays are scanned with a laser fluorescent scanner with a computer-controlled X-Y stage and a microscope objective.
• Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser and the emitted light is split by wavelength and detected with two photomultiplier tubes.
• Fluorescence laser scanning devices are described in Schena et al. (1996), supra and in other references cited herein.
• the fiber-optic bundle described by Ferguson et al., Nat. Biotechnol., Vol. 14, pp. 1681-1684 (1996) may be used to monitor mRNA abundance levels at a large number of sites simultaneously.
• Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12-bit analog to digital board.
• the scanned image is de-speckled using a graphics program, e.g., Hijaak Graphics Suite, and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site. If necessary, an experimentally determined correction for "cross talk" (or overlap) between the channels for the two fluorophores may be made.
• a ratio of the emission of the two fluorophores is preferably calculated. The ratio is independent of the absolute expression level of the cognate gene, but is useful for genes whose expression is significantly modulated by drug administration, gene deletion or any other tested event.
• the transcriptional state of a cell may be measured by other gene expression technologies known in the art.
• Several such technologies produce pools of restriction fragments of limited complexity for electrophoretic analysis, such as methods combining double restriction enzyme digestion with phasing primers [see, e.g., EP 0534858 A1 (1992), Zabeau et al.], or methods selecting restriction fragments with sites closest to a defined mRNA end [see, e.g., Prashar et al., Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 659-663 (1996)].
• cDNA pools such as by sequencing sufficient bases, e.g., 20-50 bases, in each of multiple cDNAs to identify each cDNA, or by sequencing short tags, e.g., 9-10 bases, which are generated at known positions relative to a defined mRNA end pathway pattern. See, e.g., Velculescu, Science, Vol. 270, pp.484-487 (1995). Measurement of Other Aspects
• aspects of the biological state other than the transcriptional state such as the translational state, the activity state or mixed aspects can be measured in order to obtain drug and pathway responses. Details of these embodiments are described in this section. Translational State Measurements
• Measurement of the translational state may be performed according to several methods.
• whole genome monitoring of protein i.e., the "proteome” [see Goffeau et al. (1996), supra]
• antibodies are present for a substantial fraction of the encoded proteins, or at least for those proteins relevant to testing or confirming a biological network model of interest.
• Methods for making monoclonal antibodies are well-known. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY (1988), which is incorporated in its entirety for all purposes.
• monoclonal antibodies are raised against synthetic peptide fragments designed based on genomic sequence of the cell. With such an antibody array, proteins from the cell are contacted to the array and their binding is assayed with assays known in the art.
• proteins can be separated by two-dimensional gel electrophoresis systems.
• Two-dimensional gel electrophoresis is well-known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. See, e.g., Hames et al., Gel Electrophoresis of Proteins: A Practical Approach, IRL Press, NY (1990); Shevchenko et al., Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 1440-1445 (1996); Sagliocco et al., Yeast, Vol. 12, pp. 1519-1533 (1996); Lander, Science, Vol. 274, pp.
• the resulting electropherograms can be analyzed by numerous techniques, including mass spectrometric techniques, western blotting and immunoblot analysis using polyclonal and monoclonal antibodies, and internal and /V-terminal micro-sequencing. Using these techniques, it is possible to identify a substantial fraction of all the proteins produced under given physiological conditions, including in cells, e.g., in yeast; exposed to a drug or in cells modified by, e.g., deletion or over-expression of a specific gene.
• response data may be formed of mixed aspects of the biological state of a cell.
• Response data can be constructed from, e.g., changes in certain mRNA abundances, changes in certain protein abundances, and changes in certain protein activities.
• the computation steps of the previous methods are implemented on a computer system or on one or more networked computer systems in order to provide a powerful and convenient facility for forming and testing models of biological systems.
• the computer system may be a single hardware platform comprising internal components and being linked to external components.
• the internal components of this computer system include processor element interconnected with a main memory.
• computer system can be an Intel Pentium based processor of 200 Mhz or greater clock rate and with 32 MB or more of main memory.
• the external components include mass data storage.
• This mass storage can be one or more hard disks, which are typically packaged together with the processor and memory. Typically, such hard disks provide for at least 1 GB of storage.
• Other external components include user interface device, which can be a monitor and keyboards, together with pointing device, which can be a "mouse" or other graphic input devices.
• the computer system is also linked 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.
• Loaded into memory during operation of this system are several software components, which are both standard in the art and special to the instant invention. These software components collectively cause the computer system to function according to the methods of this invention.
• the software component represents the operating system, which is responsible for managing the computer system and its network interconnections.
• This operating system can be, e.g., of the Microsoft Windows family, such as Windows 95, Windows 98 or Windows NT; or a Unix operating system, such as Sun Solaris.
• Software include common languages and functions conveniently present on this system to assist programs implementing the methods specific to this invention. Languages that can be used to program the analytic methods of this invention include C, C++ or, less preferably, JAVA.
• the methods of this invention are programmed in mathematical software packages which allow symbolic entry of equations and high-level specification of processing, including algorithms to be used, thereby freeing a user of the need to procedurally program individual equations or algorithms.
• Such packages include, e.g., Matlab from Mathworks (Natick, Mass.), Mathematica from Wolfram Research (Champaign, IL) and MathCAD from Mathsoft (Cambridge, MA).
• the analytic software component actually comprises separate software components which interact with each other.
• Analytic software represents a database containing all data necessary for the operation of the system.
• Analytic software includes a data reduction and computation component comprising one or more programs which execute the analytic methods of the invention.
• Analytic software also includes a user interface which provides a user of the computer system with control and input of test network models, and, optionally, experimental data.
• the user interface may comprise a drag-and-drop interface for specifying hypotheses to the system.
• the user interface may also comprise means for loading experimental data from the mass storage component, e.g., the hard drive; from removable media, e.g., floppy disks or CD-ROM; or from a different computer system communicating with the instant system over a network, e.g., a local area network, or a wide area communication network, such as the Internet.
• a network e.g., a local area network, or a wide area communication network, such as the Internet.
• Absolute CC values are shown for expression levels analyzed in the 18 test samples only (R1) or in all 36 samples (R2).
• Probe Set Probe set
• Probe set Probe set Name R1 R2 name R1 R2 name R1 R2 37628 at 0.865 0.808 37529 at 0.669 0.494 201 s at 0.577 0.571 41859 at 0.865 0.877 32175 at 0.669 0.486 774_g_at 0.576 0.465 38120 at 0.852 0.768 35753 at 0.667 0.535 40998 at 0.576 0.539 32664 at 0.848 0.749 38875 r at 0.667 0.495 41772 at 0.573 0.485 35717 at 0.847 0.783 32779 s at 0.665 0.599 40522 at 0.572 0.544 34257 at 0.846 0.786 41385 at 0.665 0.594 41768 at 0.571 0.513 38220 at 0.844 0.820 1319 at 0.664 0.580 37828 at 0.569 0.677 40423 at

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