WO2006124022A1 - Profilage d’expression de gene de micromatrice dans des sous-types d’hypernephrome - Google Patents

Profilage d’expression de gene de micromatrice dans des sous-types d’hypernephrome Download PDF

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WO2006124022A1
WO2006124022A1 PCT/US2005/016889 US2005016889W WO2006124022A1 WO 2006124022 A1 WO2006124022 A1 WO 2006124022A1 US 2005016889 W US2005016889 W US 2005016889W WO 2006124022 A1 WO2006124022 A1 WO 2006124022A1
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probes
rcc
protein
aggressive
genes
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PCT/US2005/016889
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Bin Tean Teh
Minhan Tan
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Vanandel Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • This invention relates to the field of molecular biology and medicine, including gene expression profiling for cancer, specifically, clear cell renal cell carcinoma.
  • RCC Renal cell carcinoma
  • CC-RCC is caused by neoplasia of proximal renal tubular epithelium; and is a prime example of a clinically heterogeneous disease for which treatment options are largely ineffective for advanced stage tumors.
  • the cancer is more common in men than women, especially men over 55 years of age. It affects approximately 3/10,000 people; 18,000 new cases arise in the U.S. annually, of which about 8,000 result in death; worldwide fatalities were estimated to exceed 100,000 in 2001.
  • CC-RCC represents 2% of all malignancies and 2% of all cancer-related deaths. Approximately 30% of patients present with metastatic disease and life expectancies averaging only nine months.
  • CC-RCC The CC-RCC tumor can be one centimeter in diameter when discovered (usually incidentally), or as bulky as several kilograms. Most often it manifests with pain, as a palpable mass or with hematuria; a variety of paraneoplastic syndromes have been described. CC-RCC may first manifest with metastases after being clinically silent for years. The characteristic gross appearance of the tumor is solid, lobulated, and yellow, with variegation due to necrosis and hemorrhage. Tumor may be well circumscribed, or may invade the perirenal adipose tissue or the renal vein. Cystic degeneration is common, though some tumors are predominantly cystic. Hartman et al, Urology 28:145- 153 (1986).
  • T, N, and M categories are determined by physical examination and imaging. Sobin, L.H. et al, eds., TNM classification of malignant tumors. 5 th ed. (John Wiley & Sons, New York 1997). This system is set forth in Table 1 below. Approximately one-third of initially diagnosed CC-RCC patients present with metastatic disease, and 40% of individuals undergoing surgical resection or radical nephrectomy will eventually develop metastasis.
  • Tumor is ⁇ 7.0 cm in greatest dimension, limited to the kidney T2 Tumor is >7.0 cm in greatest dimension, limited to the kidney T3 Tumor extends into major veins or invades adrenal or perinephric tissues but not beyond Gerota fascia
  • T3a Tumor invades adrenal gland or perinephric tissues but not beyond Gerota fascia
  • T3b Tumor grossly extends into renal vein(s) or vena cava below diaphragm
  • T3c Tumor grossly extends into vena cava above diaphragm
  • pTNM Pathological Classification corresponds to the T, N, and M categories.
  • CC-RCC may exist as two distinct subtypes: aggressive and non-aggressive.
  • the aggressive form the primary tumor grows more rapidly, tends to metastasize sooner, the metastases grow more rapidly, and the patients die sooner.
  • Patients manifesting the aggressive subtype typically manifest Stages III or rV.
  • Non-aggressive RC patients typically manifest at Stages I or II.
  • predicting individual survival in CC-RCC is challenging based on clinico-pathologic parameters.
  • an understanding of the genetic mechanisms underlying the variability in CC-RCC behavior is a key priority for oncology.
  • R. J. Motzer J Clin Oncol 21, 1193 (2003).
  • VHL von Hippel-Lindau
  • HIF hypoxia-inducible factor
  • Aurora kinase A, B and C are key mitotic regulators required for genomic stability. They are involved in multiple steps of mitosis, including centrosome duplication, formation of a bipolar mitotic spindle, chromosome alignment on the mitotic spindle, and the fidelity monitoring spindle checkpoint. While this gene has been previously studied in simpler organisms, and in other types of cancers such as breast, colon and lung cancer, overexpression of these genes has not been previously studied in kidney cancer.
  • the inventors Using a clinically well-characterized patient population, the inventors correlated the global gene expression profiling of CC-RCC with tumor progression and clinical outcome, even in the absence of known cellular or molecular characteristics of these tumors.
  • the inventors identified common features of renal cell tumorigenesis, including, genes that were upregulated when comparing aggressive CC-RCC tumors (poor prognosis) to non-aggressive CC-RCC tumors (good prognosis), thus enabling the inventors to identify specific molecular signatures of aggressive and non-aggressive subtypes of CC-RCC tumors.
  • the discovery of a set of differentially expressed genes for each subtype provides a basis for explaining the differences in aggressiveness and clinical outcome.
  • the methods and compositions described herein permit identification of proteins whose detection provide an early diagnostic approach to CC-RCC proteins as well as drug targets for the products of these genes.
  • CC-RCC cancer-specific regulatory protein
  • the methods and compositions described herein permit identification of proteins whose detection provide an early diagnostic approach to CC-RCC proteins as well as drug targets for the products of these genes.
  • a particular gene is differentially regulated in aggressive CC-RCC, one can focus on developing drugs that suppress up-regulation, act directly on the protein product, or bypass the step in a cellular pathway mediated by the product of this gene.
  • the present inventors have discovered expressed nucleic acid markers through statistical clustering analysis, namely, fully supervised principal components and unsupervised two-means clustering to derive optimal oligonucleotide predictors that yielded both continuous and discrete survival predictions.
  • the predictors were independently significant of all combinations of up to two clinical pathological parameters studied. Further, the inventors validated their studies across different microarray platforms and experimental designs.
  • the present invention provides a nucleic acid probe or set of probes (preferably between two and 266 in number) and a microarray comprising these DNA markers as probes for the gene expression levels that are characteristic of CC-RCC tumor tissue.
  • the presence and levels of mRNA in a tissue being analyzed are screened using methods known in the art (i.e., Southern/Northern/Western blotting, gel electrophoresis, RFLP, SSCP).
  • the invention is further directed to a method of implementing a microarray technology for disease prognosis (aggressive versus non- aggressive CC-RCC) thereby supplementing currently available prognostic techniques and pathological classification.
  • the present invention also is directed to a prognostic microarray composition of at least one oligonucleotide or polynucleotide probe from a set of probes immobilized to a solid surface in a predetermined order such that a row of pixels corresponds to replicates of one distinct probe from the set.
  • the probes are complementary to nucleic acid sequences expressed differentially in aggressive as compared to non-aggressive types of CC-RCC.
  • the probes are preferably any of SEQ ID NOS.: 1-266 inclusive.
  • the nucleic acid sequences hybridize to the probes under high stringency conditions.
  • the microarray may comprise at least about ten probes, or in another embodiment, at least seventy-five probes; in another embodiment at least 114 probes; in another embodiment at least fifty probes; and in another embodiment at least twenty-seven probes, which probes are complementary to nucleic acid sequences expressed differentially in aggressive as compared to non-aggressive types of CC-RCC. These probes are preferably at least about fifteen nucleotides in length.
  • the present invention also includes a kit comprising the inventive composition; means for carrying out hybridization of the nucleic acid to the probe(s); and means for reading hybridization data.
  • the kit includes the inventive microarray, reagents that facilitate hybridization of the nucleic acid to the immobilized probes, and a computer- readable storage medium comprising logic which enables a processor to read data representing detection of hybridization.
  • the present invention also includes a method for assessing the aggressiveness of
  • CC-RCC in a renal rumor tissue sample the relative expression of genes in a subject's CC-RCC tumor tissue is compared to the same genes in a population of renal tumor tissue samples.
  • the genes are selected from the group consisting of SEQ ID NOS.1-266.
  • Another method discovered by the inventors includes evaluating the aggressiveness of CC-RCC in a patient by detecting the level of expression in a renal tumor tissue sample of two or more genes from Table 7, wherein differential expression of the genes in Table 7 indicates whether the CC-RCC is aggressive or non-aggressive.
  • the genes in Table 8 are evaluated for aggressiveness of CC-RCC in a patient
  • the genes in Table 9 are evaluated for aggressiveness of CC-RCC in a patient.
  • the invention also includes a diagnostic portfolio of nucleic acid sequences selected from the group consisting of SEQ ID NOS.: 1-266. More preferably, the diagnostic profile is in a matrix suitable for identifying the differential expression of the genes contained therein.
  • the present invention provides a nucleic acid probe or a set of probes (preferably between 2 and 314 in number) and a microarray comprising these DNA markers as probes for the gene expression levels that are characteristic of CC-RCC tumor tissue compared to normal tissue from the same kidney. In one embodiment, the presence and levels of mRNA in a tissue being analyzed are screened using methods known in the art (i.e., Southern/Northern/Western blotting, gel electrophoresis, RFLP, SSCP).
  • the microarray of the present invention can be used to assay expressed nucleic acid samples (representing genes differentially expressed in normal kidney versus CC- RCC tumor tissue) for one or more individual subject's tumor or normal tissue, wherein each sample from an individual subject's tumor or normal tissue is spotted column- wise on the pixels of the microarray probes.
  • the microarray can comprise at least 10, or, in another embodiment, at least about 314 probes.
  • the differentially expressed nucleic acid sequences detected by the probes may be ones that are up-regulated or down-regulated in one form of CC-RCC compared to normal tissue or compared to the other form of CC-RCC (aggressive versus non- aggressive).
  • the above probes are typically of mammalian, preferably human, origin.
  • the nucleic acids from the tumor and the tissue are detectably labeled, preferably with a fluorescent label prior to the hybridization. With fluorescent labels, hybridization is detected as a fluorescent signal bound to the probe.
  • the probes are immobilized to a solid surface of a microarray as pixels arranged in rows, and the expressed nucleic acids from the tumor tissue or normal tissue samples are spotted column-wise onto the probe pixels.
  • a protein product of at least one gene is selected based on its expression being up-regulated in a majority of CC-RCC patients.
  • This protein product is preferably a secreted protein or a cell surface protein expressed in tissue readily accessible for assay.
  • the presence or quantity of the protein product in a body fluid or a tissue or cell sample from the subject is determined.
  • An increased level of the protein product compared to the level in a normal subject's fluid, tissue, or cells (or another reference normal value) is indicative of the presence of a CC-RCC tumor in the subject.
  • This invention also provides a method for diagnosing the recurrence of a CC-RCC tumor in a subject in whom a CC-RCC primary tumor has been excised or otherwise treated.
  • a protein product of at least one gene is selected based on its expression being up-regulated in a majority of CC-RCC patients.
  • This protein product is preferably a secreted protein or a cell surface protein expressed in tissue readily accessible for assay.
  • the presence or quantity of the protein product in a body fluid or a tissue or cell sample from the subject is determined.
  • An increase in the level of the protein product compared to the level in a normal subject's fluid, tissue, or cells (or another reference normal value) is indicative of the presence of a recurrent CC-RCC tumor in the subject.
  • the gene is preferably one that hybridizes with any one or more of SEQ ID NOS: 240-553.
  • Figures 1A-1C show good prognosis (non-aggressive) and poor prognosis
  • Figures 2A-2C show good prognosis (non-aggressive) and poor prognosis
  • Figure 3 shows images of forty-five formalin-fixed paraffin-embedded CC-RCC samples by CD 31 immunostaining in good prognosis (non-aggressive) and poor prognosis (aggressive) tumors (Figs. 3A and 3B, respectively).
  • Figure 4A shows a comparative genomic microarray analysis from gene expression ratios derived from a ninety-two tumor oligonucleotide data set and twelve oligonucleotide expression profiles of non-cancerous kidney cortical samples.
  • Figure 4B shows a comparative genomic microarray analysis inferred fom cytogenetic profiles of thirteen metastatic tumors.
  • Figure 5A is a heat map showing two distinct clusters (good prognosis and poor prognosis) formed by a hierarchical clustering of thirty-four informative cDNA clones corresponding to a reduced supervised PC predictor.
  • Figure 5B shows corresponding survival curves for the two distinct clusters.
  • Figure 6 is a heat map showing four distinct subclusters of good-prognosis tumors with a significantly high prevalence of VHL gene mutations formed by hierarchical clustering of thirty-seven transcripts corresponding to a two-means predictor.
  • Figure 7 is a heat map showing co-regulation of Aurora kinase pathway genes.
  • a can mean one or more, depending on the context with which it is used; the acronym “PCR” is used interchangeably with “polymerase chain reaction”; and the term “oligonucleotide” refers to primers, probes, and oligomer fragments.
  • nucleic acid and “polynucleotide” are interchangeable and refer to both DNA and RNA (as well as peptide nucleic acids).
  • oligonucleotide is not intended to be limited to a particular number of nucleotides and therefore overlaps with polynucleotide.
  • Probes for gene expression analysis include those comprising ribonucleotides, deoxyribonucleotides, both or their analogues as described below. They may be poly- or oligonucleotides, without limitation of length.
  • the present invention uses both oligonucleotide microarrays and cDNA microarrays to probe for, and to determine the relative expression of target genes of interest in a tissue sample of CC-RCC.
  • the term “specifically hybridize to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • stringent conditions refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequence. Stringent conditions are sequence-dependant and will be different in different circumstances. One skilled in the art knows how to select such conditions. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 degrees Celsius lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature
  • stringent conditions will be those in which the salt concentration is at least about 0.01 ato 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3.0 degrees Celsius for short probes (e.g., 10 to 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Microarrays are orderly arrangements of spatially resolved samples or probes (in the present invention oligonucleotides and cDNAs of known sequence) that allow for massively parallel gene expression and gene discovery studies (Lockhart DJ et at, Nature (2000) 405 (6788):827-836).
  • DNA microarrays are fabricated by high-speed robotics. Microarray technology adds automation to the process of resolving nucleic acids of particular identity and sequence present in an analyte sample by labeling, preferably with fluorescent labels, and subsequent hybridization to their complements immobilized to a solid support in microarray format. An experiment with a single DNA chip can provide simultaneous information on thousands of genes- a dramatic increase in throughput (Reichert et ah (2000) Anal. Chem. 72:6025-6029) when compared to traditional methods.
  • Array experiments employ common solid supports such as glass slides, microplates or standard blotting membranes, and can be created by photolithographic synthesis by robotic deposition of samples.
  • Photolithography generally involves attaching synthetic linkers (modified with photochemically removable protecting groups) to a glass substrate and directing light through a photolithographic mask to deprotect specific areas on the surface. The first of a series of hydroxyl-protected deoxynucleotides is incubated with the surface, and chemical binding occurs at the sites previously illuminated. Using a new mask, light then is directed to different regions of the substrate, and the chemical cycle repeated.
  • Probes may be synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. Sample spot sizes in microarrays are typically ⁇ 200 ⁇ m in diameter, and these arrays usually contain thousands of spots.
  • Microarrays require specialized robotics and imaging equipment that generally are commercially available and well-known in the art.
  • Microarray analysis generally involves injecting a fluorescently tagged nucleic acid sample into a chamber to hybridize with complementary oligonucleotides on the microarray slide; laser excitation at the interface of the array surface and the tagged sample; collection of fluorescence emission by a lens; optical filtration of the fluorescence emissions; fluorescence detection; and quantification of hybridization intensity.
  • Oligonucleotide arrays are based on sequence information and are targeted to monitor the expression levels of many genes. Using as little as 200 to 300 bases of a gene, cDNA, or EST sequence, independent 25-mer oligonucleotides are selected (non- overlapping or minimally overlapping) as detectors. Probe selection is based upon several factors: complementarity of the probe to a selected gene, cDNA, or EST sequence; uniqueness relative to family members and other genes; and an absence of near-complementarity to other common RNAs that may be in the sample.
  • probe redundancy i.e., using multiple oligonucleotides having different sequences but designed to hybridize to different regions of the same RNA.
  • additional redundancy involves the use of "mismatch control probes” that are identical to their "perfect match” partners except for a single base difference in a central position. Even with low concentrations of RNA, hybridization to the perfect match/mismatch pairs yields identifiable fluorescence patterns. The strength of these patterns indicates the concentration of the RNA in the sample. Lipshutz, Robert J., Fodor, Stephen P.A., Gingeras, Thomas R., Lockhart, David J. High density synthetic oligonucleotide arrays. Nature Genetics Supplement. 21:20-23 (1999).
  • Format II an array of probes that are "natural" oligo- or polynucleotides
  • oligomers of 20 ⁇ 80 bases oligonucleotide analogues e.g., with phosphorothioate, methylphosphonate, phosphoramidate, or 3'-aminopropyl backbones
  • PNA peptide-nucleic acids
  • the array is (1) exposed to an analyte comprising a detectable labeled, preferably fluorescent, sample nucleic acid (typically DNA), (2) allowed to hybridize, and (3) the identity and/or abundance of complementary sequences is determined.
  • a detectable labeled preferably fluorescent, sample nucleic acid (typically DNA)
  • the preferred analyte of this invention is isolated from tissue biopsies before they are stored or from fresh-frozen tumor tissue of the primary tumor which may be stored and/or cultured in standard culture media.
  • total RNA or poly(A)-containing mRNA is isolated using commercially available reagents and kits, e.g., from Invitrogen, Oligotex, or Qiagen.
  • the mRNA is reverse transcribed into cDNA in the presence of labeled nucleotides.
  • cDNA is generally synthesized using reverse transcriptase (e.g., Superscript II reverse-transcription kit from GEBCO-BRL). This may be directly or indirectly labeled by conjugation with a fluorescent dye.
  • the materials for a particular application of microarray technology are not necessarily available in convenient in kit form.
  • the present invention provides microarrays and kits useful for analysis and prognosis of CC-RCC samples.
  • the present invention includes microarrays comprising one or more nucleic acid probes having hybridizable fragments of any length (from about 15 bases to full coding sequence) for the genes whose expression is to be analyzed.
  • the full length sequence must not necessarily be known, as those of skill in the art will know how to obtain the full length sequences using the sequence of a given EST and known data mining, bioinformatics, and DNA sequencing methodologies without undue experimentation.
  • the probe of choice for a particular gene can be the full length coding sequence or any fragment thereof having at least about 15 nucleotides.
  • the practitioner can select any appropriate fragment of that sequence.
  • partial sequence information e.g., an EST probe
  • the full length sequence of which that EST is a fragment becomes available e.g., in a genome database
  • the skilled artisan can select a longer fragment than the initial EST, as long as the length is at least about 15 nucleotides.
  • the polynucleotide or oligonucleotide probes of the present invention may be native DNA or RNA molecules or analogues of DNA or RNA or portions thereof.
  • the present invention is not limited to the use of any particular DNA or RNA analogue or portion thereof; rather any one is useful provided that it is capable of adequate hybridization to the complementary DNA (or mRNA) in a test sample, has adequate resistance to nucleases and stability in the hybridization protocols employed.
  • DNA or RNA may be made more resistant to nuclease degradation in vivo by modifying internucleosite linkages (e.g., methylphosphonates or phosphorothioates) or by incorporating modified nucleosides (e.g., 2'-0-methylribose or l'- ⁇ -anomers) as described below.
  • internucleosite linkages e.g., methylphosphonates or phosphorothioates
  • modified nucleosides e.g., 2'-0-methylribose or l'- ⁇ -anomers
  • a poly- or oligonucleotide may comprise at least one modified base moiety, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl- ⁇ -thiouridine, 5-carboxymethyl-aminomethyl uracil, dihydrouracil, ⁇ -D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5 -methoxyamino-methyl-2-thiouracil, ⁇ -D-mannosylqueosine, 5-methoxy-carboxymethyluracil, 5-methoxyuracil, 5-me
  • the poly- or oligonucleotide may comprise at least one modified sugar moiety including, but not limited, to arabinose, 2-flourarabinose, xylulose, and hexose.
  • the poly- or oligonucleotide probe comprises a modified phosphate backbone synthesized from a nucleotide having, for example, one of the following structures: a phosphorothioate, a phosphoridothioate, a phosphoramidothioate, a phosphoramidate, a phosphordiimidate, a methylsphosphonate, an alkyl phosphotriester, 3'-aminopropyl and a formacetal or analog thereof.
  • the poly- or oligonucleotide probe is an ⁇ -anomeric oligonucleotide which forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al,, 1987, Nucl. Acids Res. 15:6625-6641).
  • An oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a hybridization-triggered cleavage agent, etc., all of which are well-known in the art.
  • Oligonucleotides of this 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.).
  • 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. (1998) 16:3209
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymet supports (Sarin et al, Proc. Natl. Acad. ScL USA (1988) 85:7448-7451), etc.
  • Preferred detectable labels include radionuclides, fluorescers, fluourogens, a chromophore, a chromogen, a phosphoescer, a chemiluminescer or a bioluminescer.
  • fluorescers or fluorogens are i fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, a fluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green or Texas Red.
  • Common fluorescent labels include fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Most preferred are the labels described in the Examples, below.
  • the fluorophore must be excited by light of a particular wavelength to flouresce.
  • Fluorescein, fluorescein derivatives and fluorescein-like molecules such as
  • Rhodamine GreenTM and Rhodol GreenTM are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl- reactive groups.
  • fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziridine-reactive groups.
  • the long wavelength rhodamines which are basically Rhodamine GreenTM derivatives with substituents on the nitrogens, are among the most photostable fluorescent labeling reagents known. Their spectra are not affected by changes in pH between 4 and 10, an important advantage over the fluouresceins for many biological applications.
  • This group includes the tetramethylrhodamines, X-rhodamines and Texas RedTM derivatives.
  • Other preferred fluorophores are those which are excited by ultraviolet light. Examples include cascade blue, coumarm derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives.
  • the present invention serves as a basis for even broader implementation of microarrays and gene expression in deducing critical pathways implicated in cancer, hi the case of CC-RCC, which is the focus of the present invention, a database of known patient genetic profiles can be used to categorize each new CC-RCC patient.
  • the gene expression profile of the newly diagnosed CC-RCC patient is compared to the known CC-RCC molecular database of patients, such as that described herein based on 110 patients in whom complete clinical follow up information is available,. This database will grow with each patient who is subjected to the present analysis as soon as his clinical outcome information becomes available.
  • the newly diagnosed patient's gene expression profile most closely resembles the profile of aggressive CC-RCC, as described herein, that patient will be so classified and treated accordingly, i.e., with more aggressive measures.
  • a newly diagnosed patient's profile is that of the non- aggressive type, he will be treated accordingly, e.g., with less aggressive measures and careful clinical follow-up.
  • composition of the present invention may be used in diagnostic, prognostic, or research procedures in conjunction with any appropriate cell, tissue, organ or biological sample of the desired animal species.
  • biological sample any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus and the like.
  • organ or tissue extract and a culture fluid in which any cells or tissue preparation from the subject has been incubated is included within the meaning of this term.
  • the molecular profiling information described herein is also harnessed for the purpose of discovering drugs that are selected for their ability to correct or bypass the molecular alterations or derangements that are characteristic of CC-RCC, particularly those that are associated with its aggressive form. A number of approaches are available.
  • CC-RCC cell lines are prepared from tumors using standard methods and are profiled using the present methods. Preferred cell lines are those that maintain the expression profile of the primary tumor from which they were derived. One or several CC-RCC cells lines may be used as a "general" panel; alternatively or additionally, cell lines from individual patients may be prepared and used. These cell lines are used to screen compounds, preferably by high-throughput screening (HTS) methods, for their ability to alter the expression of selected genes. Typically, small molecule libraries available from various commercial sources are tested by HTS protocols.
  • HTS high-throughput screening
  • the molecular alterations in the cell line cells can be measured at the mRNA level
  • EIA enzyme immunoassays
  • RIA radioimmunoassay
  • ELISA immunofluorescence microscopy
  • antisense RNAs or DNAs that specifically inhibit the transcription and/or translation of the targeted genes can be screened for specificity and efficacy using the present methods.
  • Antisense compositions would be particularly useful for treating tumors in which a particular gene is up-regulated (e.g., the genes in Tables 2 and 3).
  • Table 12 in Examples are targets for ealy diagnostic assays of CC-RCC if the proteins can be detected by some assay means, e.g., immunoassay, in some accessible body fluid or tissue.
  • the most useful diagnostic targets are secreted proteins which reach a measurable level in a body fluid before the tumor presents by other criteria discussed in the Background section.
  • a sample of a body fluid such as plasma, serum, urine, saliva, cerebrospinal fluid, et cetera, is obtained from the subject being screened.
  • the sample is subject to any known assay for the protein analyte.
  • cells expressing the protein on their surface may be obtained, e.g., blood cells, by simple, conventional means. If the protein is a receptor or other cell surface structure, it can be detected and quantified by well-known methods such as flow cytometry, immunofluorescence, immunocytochemistry or immunohistochemistry, and the like.
  • an antibody or other protein or peptide ligand for the target protein to be detected is used.
  • the gene product is a receptor
  • a peptidic or small molecule ligand for the receptor may be used in known assays as the basis for detection and quantitation.
  • antibodies may also be used for diagnosis and prognosis, for example to image occult metastatic foci or for other types of in situ evaluations. These methods utilize include various radiographic, scintigraphic, and other imaging methods well-known in the art (MRI, PET, et cetera).
  • Suitable detectable labels include radioactive, fluorescent, fluorogenic, chromogenic, or other chemical labels.
  • Useful radiolabels, which are detected simply by gamma counter, scintillation counter, or autoradiography include 3 H, 125 I, 131 I 5 35 S, and 14 C.
  • Common fluorescent labels include fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, ⁇ -phthaldehyde and fluorescamine.
  • the fluorophore such as the dansyl group, must be excited by light of a particular wavelength to fluoresce. See, Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR, 1996.
  • Fluorescein, fluorescein derivatives and fluorescein-like molecules such as Oregon GreenTM and its derivatives, Rhodamine GreenTM and Rhodol GreenTM, are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl-reactive groups. Fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziridine-reactive groups. The long wavelength rhodamines include the tetramethykhodamines, X-rhodamines and Texas RedTM derivatives. Other preferred fluorophores for derivatizing the protein binding partner are those which are excited by ultraviolet light. Examples include cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives.
  • the protein can also be labeled for detection using fluorescence-emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the protein using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • radionuclides may be bound to protein either directly or indirectly using a chelating agent such as DTPA and EDTA which is chemically conjugated, coupled, or bound (which terms are used interchangeably) to the protein. The chemistry of chelation is well known in the art.
  • the key limiting factor on the chemistry of coupling is that the antibody or ligand must retain its ability to bind the target protein.
  • a number of references disclose methods and compositions for complexing metals to macromolecules including description or useful chelating agents.
  • the metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules. See, for example, U.S. 5,627,286, U.S. 5,618,513, U.S. 5,567,408, U.S. 5,443,816, U.S. 5,561,220, all of which are incorporated by reference herein.
  • radionuclide having diagnostic (or therapeutic value) can be used.
  • the radionuclide is a ⁇ -emitting or a ⁇ -emitting radionuclides, for example, one selected from the lanthanide or actinide series of the elements.
  • Positron- emitting radionuclides e.g., 68 Ga or 64 Cu, may also be used.
  • Suitable ⁇ -emitting radionuclides include those which are useful in diagnostic imaging applications.
  • the gamma-emitting radionuclides preferably have a half-life of from one hour to forty days, preferably from twelve hours to three days.
  • Suitable ⁇ -emitting radionuclides include 67 Ga, 111 In, 99m Tc, 169 Yb and 186 Re.
  • suitable radionuclides are 67 Cu, 67 Ga, 68 Ga, 72 As, 89 Zr, 90 Y, 97 Ru, 99 Tc, 111 In 5 123 1, 125 1, 131 1, 169 Yb, 186 Re, and 201 Tl.
  • positron-emitting radiometals as labels
  • certain proteins such as transferrin and human serum albumin, have been labeled with 68 Ga.
  • a number of metals (not radioisotopes) useful for MRI include gadolinium, manganese, copper, iron, gold, and europium. Gadolinium is most preferred. Dosage can vary from 0.01 mg/kg to 100 mg/kg.
  • In situ detection of the labeled protein may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label.
  • histological methods such as staining procedures
  • An alternative diagnostic approach utilizes cDNA probes that are complementary to and thereby detect cells in which a gene associated with CC-RCC is upregulated by in situ hybridization with mRNA in these cells.
  • the present invention provides methods for localizing target mRNA in cells using fluorescent in situ hybridization (FISH) with labeled cDNA probes having a sequence that hybridizes with the mRNA of an upregulated gene.
  • FISH fluorescent in situ hybridization
  • the basic principle of FISH is that DNA or RNA in the prepared specimens are hybridized with the probe nucleic acid that is labeled non-isotopically with, for example, a fluorescent die, biotin, or digoxigenin.
  • the hybridized signals are then detected by fluorimetric or by enzymatic methods, for example, by using a fluorescence or light microscope. The detected signal and image can be recorded on light sensitive film.
  • An advantage of using a fluorescent probe is that the hybridized image can be readily analyzed using a powerful confocal microscope or an appropriate image analysis system with a charge-coupled device (CCD) camera.
  • CCD charge-coupled device
  • FISH offers increased sensitivity. In addition to offering positional information, FISH allows better observation of cell or tissue morphology. Because of the nonradioactive approach, FISH has become widely used for localization of specific DNA or mRNA in a specific cell or tissue type.
  • the coding DNA or portions thereof can be expression-cloned to produce a polypeptide or peptide epitope thereof. That protein or peptide can be used as an immunogen to immunize animals for the production of antisera or to prepare monoclonal antibodies (mAbs). These polyclonal sera or mAbs can then be applied in an immunoassay, preferably an EIA, to detect the presence of protein Y or measure its concentration in a body fluid or cell/tissue sample.
  • an immunoassay preferably an EIA
  • an agonist or mimetic would be administered to maximize binding and activation of those receptor molecules which are expressed.
  • a therapy targeted specifically at this form of the cancer that would be used alone or in combination with known therapeutic approaches as discussed above.
  • a preferred approach would be to stimulate production of the protein by administering an agent that promoted production, enhanced its stability or inhibited its degradation or metabolism.
  • gene therapy methods could be used to introduce more copies of the affected gene or more actively expressed genes operatively linked to strong promoters, e.g., inducible promoters, such as an estrogen inducible system.
  • strong promoters e.g., inducible promoters, such as an estrogen inducible system.
  • inducible promoters such as an estrogen inducible system.
  • repressible systems driven by the conventional antibiotic, tetracycline. Gossen, M. et al, Proc. Natl. Acad. ScL USA 89:5547-5551 (1992).
  • Antibodies may be administered to a patient to bind and inactivate (or compete with) secreted protein products or expressed cell surface products or upregulated genes.
  • gene therapy methods could be used to introduce antisense oligonucleotide or polynucleotide constructs that would inhibit gene expression in a highly specific manner.
  • Such constructs could be operatively linked to strong promoters, e.g., inducible promoters, such as an estrogen inducible system (Braselmann, S. et al,. Proc. Natl. Acad. Sd. USA (1993) 90:1657-1661).
  • inducible promoters such as an estrogen inducible system
  • repressible systems driven by the conventional antibiotic, tetracycline (Gossen, M. et al, Proc. Natl. Acad. ScL USA 89:5547-5551 (1992)).
  • antisense constructs specific for different upregulated genes could be employed together.
  • the sequences of the upregulated genes described herein are used to design the antisense oligonucleotides (Hambor, J.E. et al., J. Exp. Med. 168:1237-1245 (1988); Holt, J.T. et al., Proc. Natl Acad. ScL 83:4794-4798 (1986); Izant LG. et al., Cell 36:1007-1015 (1984); Izant, LG. et al, Science 229:345- 352 (1985); De Benedetti, A. et al, Proc. Natl. Acad. ScL USA 84:658-662 (1987)).
  • the antisense oligonucleotides may range from 6 to 50 nucleotides, and may be as large as
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, sing-stranded or double-stranded.
  • the oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone (as discussed above).
  • the oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see., e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. USA 84:684-652; PCT Publication No.
  • WO 88/09810 published December 15, 1988
  • blood-brain barrier e.g., PCT Publication No. WO 89/10134, published April 25, 1988
  • hybridization-triggered cleavage agents e.g., Krol et al, 1988, BioTechniques 6:958-97
  • intercalating agents e.g., Zon, 1988, Pharm. Res. 5:539-549.
  • the therapeutic methods that require gene transfer and targeting may include virus-mediated gene transfer, for example, with retroviruses (Nabel, E.G. et al, Science 244:1342 (1989), Antiviruses, and recombinant adenovirus vectors (Horowitz, M.S., In: Virology, Fields, B.N. et al, eds. Raven Press, New York, 1990, p. 1679, or current edition; Berkner, K.L., Biotechniques 6:616-919, 1988), Straus, S.E., In: The Adenoviruses, Ginsberg, H.S., ed., Plenum Press, New York, 1984, or current edition).
  • Adeno-associated virus also is also useful for human gene therapy (Samulski, RJ., et al., EMBO J. 10:3941 (1991); Lebkowskie, J.S. et al, MoI. Cell Biol. (1988) 8:3988-3996; Kotin, R.M. et al., Proc. Natl. Acad. Sci. USA (1990) 87:2211-2215; Hermonat, PL et al, J. Virol. (1984) 51: 329-339). Improved efficiency is attained by the used of promoter enhancer elements in the plasmid DNA constructs (Philip R., et al, J. Biol. Chem. (1993) 268:16087-16090).
  • Preferred carriers are targeted liposomes (Nicolau, C. et al, Proc. Natl. Acad. Sci.
  • Liposomes have been used to encapsulate and deliver a variety of materials to cells, including nucleic acids and viral particles (Faller, D. V. et al, J. Virol. (1984) 49:269-272).
  • Preformed liposomes that contain synthetic cationic lipids form stable complexes with polyanionic DNA (Feigner, P.L. et al, Proc. Natl. Acad. Sci. USA (1987) 84:7413- 7417).
  • Cationic liposomes, liposomes comprising some cationic lipid, that contained a membrane fusion-promoting lipid dioctadecyldimethyl-ammonium-bromide (DDAB) have efficiently transferred heterologous genes into eukaryotic cells (Rose, J.K. et al, Biotechniques (1991) 10:520-525).
  • Cationic liposomes can mediate high level cellular expression of transgenes, or mRNA, by delivering them into a variety of cultured cell lines (Malone, R., et al, Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081).
  • expression level can be detected by reverse transcription PCR if the the sample DNA is in formalin-fixed paraffin-embedded tissue.
  • Example 1- Patients and tumor samples
  • the total percentage count for each category may not total to 100 due to rounding.
  • a total of 146 primary tumor and thirteen metastatic tissue specimens from independent patients were received from eight centers (six in the U.S.A., two in Japan). Each tumor sample was grossly dissected, flash frozen following resection and stored at - 80 0 C at each center until transport. Thirty-six of 146 primary tumor samples were degraded on quality assessment of RNA by denaturing gel electrophoresis. Baseline characteristics of viable and non- viable tissue did not significantly differ (Table 2).
  • CC-RCC clear cell renal cell carcinoma
  • AU samples had at least 60% tumor tissue.
  • Tumor staging and grading were obtained from review of the pathology reports and evaluation of the case notes by individual clinicians. Node status was either assessed intraoperatively by inspection or by pathological evaluation. Tumors with sarcomatoid change were classified as grade four tumors.
  • ECOG Eastern Cooperative Oncology Group
  • RNEasy kit Qiagen, CA.
  • the HGUl 33 Plus 2.0 GeneChips contains 54,675 probe sets, representing approximately 47,000 transcripts and variants. 5 - 20 ⁇ g of total RNA was used to prepare antisense biotinylated RNA.
  • GCOS GeneChip Operating System
  • the default unsupervised analysis approach was hierarchical clustering using complete linkage clustering with a Euclidean distance metric.
  • the default supervised analysis approach was prediction analysis of microarrays (PAM) with ten-fold cross validation over thirty thresholds and an offset percentage of 50%.
  • PAM microarrays
  • the default significance threshold was 0.05.
  • the robust multichip average (RMA) algorithm was the default method used to perform pre-processing of the CEL files, including background adjustment, quantile normalization and summarization.
  • PC principal components
  • the inventors used ten-fold cross-validation over fifty Cox score thresholds to obtain an optimal significant threshold for the Cox scores of each gene within the training set.
  • This threshold was 2.540206.
  • the first principal component of genes with absolute Cox scores exceeding that threshold was used as a gene predictor of continuous outcome; discrete classification was obtained by dividing the outcomes in two groups.
  • the inventors computed an importance score for each feature equal to its correlation with the supervised PC predictor. Shrinkage over sixty thresholds was performed, and the maximal shrinkage threshold corresponding to a significant likelihood ratio was selected. This corresponded to 77 transcripts. Validation of this reduced signature was performed on the test set.
  • a two-means clustering predictor was generated by the initial application of two- means clustering on the training set, followed by the derivation of a shrunken gene predictor with a minimum misclassification error by PAM. Multiple thresholds with a minimum misclassification error of 3% were noted, corresponding to 1, 3, 5, 12, 22, 37, 3733, 5412 and 11809 transcripts.
  • the inventors thus selected the thirty-seven transcript predictor as the most conservative shrinkage threshold within the former group of thresholds.
  • UISS UCLA Integrated Staging System
  • the UISS is an externally validated aggregate variable composed of tumor stage, grade and ECOG performance status (A. Zisman et al, J Clin Oncol 19, 1649 (2001); A. Zisman, A. J. Pantuck, R. A. Figlin, A. S. Belldegrun, J Clin Oncol 19, 3792 (2001)).
  • the inventors avoided underpowered Cox proportional hazards models by entering a maximum of three variables (corresponding to one variable per ten outcomes of interest) into the model.
  • the inventors adjusted for all thirty-six possible combinations of two variables drawn from the nine available variables (Table 4).
  • ECOG PS Eastern Cooperative Oncology Group Performance Status
  • VHL von Hippel-Lindau gene mutation status
  • UISS UCLA Integrated Staging System
  • spotted cDNA microarray gene profiling forty-two additional sample pairs of patient-matched tumor and non-cancerous cortical samples were profiled (twenty-four biological replicates firom the training set, and eighteen independent samples). A separate analysis of twenty-eight of these spotted arrays has been previously reported, together with the protocols involved. M. Takahashi et al, Proc. Natl. Acad. Sd. U S A 98, 9754 (2001). Microarray experiments were performed with spotted cDNA arrays from different print runs with a total of 21,103 different cDNA clones. 2 ⁇ g of messenger RNA from each kidney tumor and patient-matched normal kidney tissue were used in a direct- labeling protocol for preparation of Cy5- and Cy3 cDNA probe.
  • Cytogenetic profiles of the ninety-two tumor oligonucleotide data set were inferred by identifying regional expression biases from gene expression ratios derived from the ninety-two-tumor oligonucleotide data set and twelve oligonucleotide expression profiles of non-cancerous kidney cortical samples (Fig. 4A).
  • Each block corresponding to a single chromosome represents the chromosomal expression profiles of a group of samples, and each sample is represented by a single vertical line in each block. Samples have been arranged from left to right within each block by progressively worsening prognosis as predicted in a continuous fashion by the reduced supervised PC predictor.
  • This technique identified distinct regional expression biases corresponding to common cytogenetic abnormalities in CC-RCC (G. Kovacs et al, Proc. Natl. Acad. Sd. USA S5, 1571 (1988)), such as deletions of chromosomes 3p, 6q, 9pq and 14q, as well as amplifications of chromosomes 1, 3q, 5q, 8q and 12.
  • the profiles were consistent with previous reports of associations between deletions of 9pq and 14q and poor prognosis.
  • A. M. Meloni-Ehrig Am. J. Med. Genet. 115, 164 (2002).
  • Other known cytogenetic aberrations not previously linked to survival such as amplification of Iq, 3q and deletion of 6q, were highlighted as being of potential prognostic value.
  • a sliding window algorithm is applied to each ordered gene expression subset such that within each window span a binomial test is applied under the assumption that the probability (p) of the appearance of a positive relative gene expression value equals the probability (q) of the appearance of a negative expression value.
  • n denotes the number of spans.
  • t denotes the number of non-zero and r the number of positive values within the span ⁇ g h gk ⁇ u ⁇ gk ⁇ -i-i ⁇ -
  • xy is tapered wheny ⁇ i such
  • Example 5- Oligonucleotide predictors The inventors noted from inspection of the predictors (Tables 6-9) that high expression of genes classically involved in angiogenesis, hypoxia-response and endothelial cell formation optimally predicted longer patient survival. Identified genes included vascular endothelial growth factor (VEGF), endothelial PAS domain protein 1/ hypoxia inducible factor-2 (HIF-2), kinase insert domain receptor/ VEGF receptor 2, and many others.
  • VEGF vascular endothelial growth factor
  • HIF-2 hypoxia inducible factor-2
  • kinase insert domain receptor/ VEGF receptor 2 kinase insert domain receptor/ VEGF receptor 2
  • Pathway analysis was performed on the reduced supervised PC predictor with a curated database. A single key network of gene interaction was identified and corresponding canonical signal pathways were ranked by the number of nodes involved (Table 10). The VEGF canonical signaling pathway, predominantly activated in good- prognosis tumors, was the highest ranked pathway distinguishing good- from poor- prognosis tumors.
  • the reduced supervised PC analysis was analyzed because correlation with the full supervised PC predictor is likely to select for coexpressed genes that are more biologically more coherent. Importance scores were used to score the genes for pathway analysis.
  • the Ingenuity Pathways database Summer 2004 build was used for analysis. Thirty genes were identified from the 77 transcripts as being eligible for generating networks. 73 of 77 transcripts were mapped to a known gene. Duplicate transcripts corresponding to a single gene were recorded.
  • the Ingenuity pathways analysis software identified a single key network with a score of 35, with eighteen focus genes involved. In comparison, all other identified networks had a score of two or less, with only one focus gene each. Canonical pathways in this network were ranked by the number of nodes included in this network (Table 10).
  • the Aurora Kinase pathway group of genes are of potential use as therapeutic targets in renal cell carcinoma. This therapy may take a form of pharmacological or genetic inhibition of the pathway. Pharmacological inhibition may include, but is not limited to, small molecule inhibitors, monoclonal antibodies, and other forms of targeted therapies. Genetic inhibition may include, but is not limited to, include techniques such as RNA inhibition (RNAi) or the expression of dominant-negative mutants.
  • RNAi RNA inhibition
  • MVD mean microvessel density
  • CD31 is an endothelium specific transmembrane protein expressed in vascular and lymphatic endothelium, and is a commonly used standard for assessment of microvessel density (MVD).
  • Automated CD31 immunostaining was performed on 45 formalin-fixed paraffin embedded tissue specimens with pepsin pre-digestion, and with an incubation of mouse anti-human monoclonal antibody, clone JC/70A (M0823) at a concentration of 1:25.
  • the Envision system (DAKO, Denmark) was used for detection. One slide was stained per specimen.
  • the inventors used a strategy optimized to maximize correlation between mRNA expression in bulk tissue and the immunohistochemical staining.
  • Nine fields for each slide were captured at a magnification of x 200 in a 3 x 3 arrangement with the widest possible distribution across the slide.
  • Digital image capture was performed with a Spot Insight Camera on a Nikon Eclipse E600, and each field was 0.2596 mm 2 in size.
  • Interactive image analysis was performed using Cytometrix, an in-house program for quantitative analysis of histologic images. Immunostaining, image capture and analysis were performed independently by blinded individuals. CD31 immunostained plasma cells were eliminated from the analysis.
  • VHL protein is a key regulator of the HIF pathway
  • VHL mutation was associated with survival or gene expression patterns that could further define its role in angiogenesis and hypoxia-response.
  • a mutation prevalence of 34% was determined in 97 primary tumors.
  • VHL-mutant CC-RCC tumors may be heterogeneous in gene expression. Since VHL is a mediator of the HIF signaling pathway, and many genes of the predictors are linked to HIF-signaling and angiogenesis, the inventors evaluated the possibility that distinct subclusters within the major prognostic tumor groups might be characterized by high incidences of VHL mutations.
  • the inventors pursued a modified class-discovery strategy, performing hierarchical clustering on expression values of transcripts corresponding to the two-means clustering predictor to discover subclusters with varying degrees of expression of the prognostic genes (Fig. 6).
  • Two major clusters were noted: one cluster was a poor prognosis cluster (aggressive tumors); the other cluster demonstrated three distinct subclusters, two of which were good prognosis subclusters (non-aggressive clusters), and one of which was a poor prognosis subcluster.
  • the heatmap demonstrated distinct patterns of expression corresponding to each subcluster, with progressive gradations of gene expression in the four groups (Fig. 6).
  • the majority of prognostic genes identified in the predictor was upexpressed in good- prognosis tumors (34/37) and many of these genes are angiogenesis-linked.
  • the VHL mutant-predominant subcluster had lower expression of these genes relative to the other good-prognosis subcluster, and higher expression of the same genes relative to the two poor prognosis subclusters.
  • the VHL mutations characterizing this subcluster were not distinctive. This interesting finding requires validation in an external set, and should be considered preliminary.
  • the inventors applied the predictors from the primary tumor data set to an oligonucleotide data set of metastatic CC-RCC samples (n 13) resected from a variety of distant sites after relapse, and obtained good survival predictions (Figs. 2A, 2B, 2C, Table 3).
  • CGMA of these profiles yielded similar regional gene expression biases linked to poor-prognosis (aggressive tumors), including downregulation of 9pq and 14q (Fig. 4B).
  • the inventors obtained a group of 830 transcripts that discriminated between CC-
  • RCC tumor tissue and normal noncancerous tumor tissue and defined this group using significance analysis of microarrays (two class unpaired analysis of 10,000 permutations).
  • the inventors found 830 genes with a false discovery rate of 0.01 at a delta cutoff of 10.
  • the inventors selected to top 50 up- and top 50 down-regulated transcripts ordered by magnitude of fold-change (Table 12 and Table 13, respectively) and by magnitude of d. value (a modified tstatistic) (Table 14 and Table 15, respectively).
  • the identified genes may serve as a means for therapeutic inhibition by chemical or genetics means, resulting in a novel therapy.
  • the individual detection of these genes in formalin-fixed paraffin-embedded tissue using methods such as real-time reverse transcription PCR or microarrays may serve as a means of diagnosis.
  • their detection using microarrays may serve as a means of diagnosis.
  • the gene products (taken from serum, urine, saliva, or other abundant body fluid rather than kidney tissue) of the up-regulated expressed nucleic acids can be assayed using in immunoassays known in the art (i.e., ELISA, immunocytochemistry, sandwich assays, et cetera) for the purpose of diagnosing patients with CC-RCC but do not discriminate between the heterogeneous disease severity.
  • immunoassays known in the art i.e., ELISA, immunocytochemistry, sandwich assays, et cetera

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Abstract

Sonde d’acide nucléique ou ensemble original de telles sondes dans une micromatrice. La sonde ou l’ensemble de sondes sont utiles dans le pronostic de patients avec un hypernéphrome (CC-RCC), les types de tumeurs CC-RCC agressives et non agressives étant caractérisés au moyen de profils d’expression différentielle de gènes qui s’hybrident avec une ou plusieurs de ces sondes. Des micromatrices et des nécessaires pour réaliser le profilage d’expression d’une tumeur et d’un tissu normal et des procédés pour leur utilisation sont également décrits.
PCT/US2005/016889 2005-05-13 2005-05-13 Profilage d’expression de gene de micromatrice dans des sous-types d’hypernephrome WO2006124022A1 (fr)

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US20100151588A1 (en) * 2006-04-24 2010-06-17 Peter Angel Means and methods for diagnosing and treating cancer based on the frmd3 gene
JP2016174600A (ja) * 2010-01-11 2016-10-06 ジェノミック ヘルス， インコーポレイテッド 遺伝子発現を用いて腎癌の臨床的結果の見込みを判定する方法
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JP2013516195A (ja) * 2010-01-11 2013-05-13 ジェノミック ヘルス， インコーポレイテッド 遺伝子発現を用いて腎癌の臨床的結果の見込みを判定する方法
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EP2524055A2 (fr) * 2010-01-11 2012-11-21 Genomic Health, Inc. Méthode d'utilisation d'une expression génique pour déterminer la probabilité du résultat clinique d'un cancer des reins
WO2011085263A2 (fr) 2010-01-11 2011-07-14 Genomic Health, Inc. Méthode d'utilisation d'une expression génique pour déterminer la probabilité du résultat clinique d'un cancer des reins
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JP2015143276A (ja) * 2010-04-09 2015-08-06 アムジェン インコーポレイテッド Ｂｔｎｌ９タンパク質、核酸および抗体ならびにそれらの使用
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