WO2006112842A9 - Profilage d'expression genique par microreseau dans des classes de carcinome cellulaire renal papillaire - Google Patents

Profilage d'expression genique par microreseau dans des classes de carcinome cellulaire renal papillaire

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Publication number
WO2006112842A9
WO2006112842A9 PCT/US2005/013260 US2005013260W WO2006112842A9 WO 2006112842 A9 WO2006112842 A9 WO 2006112842A9 US 2005013260 W US2005013260 W US 2005013260W WO 2006112842 A9 WO2006112842 A9 WO 2006112842A9
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prcc
probes
aggressive
genes
microarray
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PCT/US2005/013260
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English (en)
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WO2006112842A3 (fr
WO2006112842A8 (fr
WO2006112842A2 (fr
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Bin Tean Teh
Minhan Tan
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Vanandel Res Inst
Bin Tean Teh
Minhan Tan
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Priority to PCT/US2005/013260 priority Critical patent/WO2006112842A2/fr
Priority to PCT/US2005/020840 priority patent/WO2006112867A2/fr
Publication of WO2006112842A2 publication Critical patent/WO2006112842A2/fr
Publication of WO2006112842A9 publication Critical patent/WO2006112842A9/fr
Publication of WO2006112842A8 publication Critical patent/WO2006112842A8/fr
Publication of WO2006112842A3 publication Critical patent/WO2006112842A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • 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

Definitions

  • This invention relates to the field of molecular biology and medicine, including gene expression profiling for cancer, specifically, papillary renal cell carcinoma.
  • Kidney cancer is a heterogenous disease consisting of various subtypes with diverse genetic, biochemical, and morphologic features.
  • Epithelial renal cell carcinoma (RCC) accounts for the vast majority of renal malignancies in adults. Renal cell carcinoma is the tenth most common cancer in the United States. A. Jemal et al, CA Cancer J Clin 54, 8 (2004).
  • RCC can be divided into clear cell (conventional), papillary (chromophil), chromophobe, collecting duct and unclassified subtypes.
  • Papillary RCC is the second most common subtype comprising 10-15% of kidney cancers (Kovacs, G., M. Akhtar, BJ. Beckwith, P. Bugert, CS. Cooper, B. Delahunt, J.N. EbIe, S. Fleming, B. Ljungberg, LJ. Medeiros, H. Moch, V.E. Reuter, E. Ritz, G. Roos, D. Schmidt, J.R. Srigley, S. Storkel, E. van den Berg, and B. Zbar, The Heidelberg classification of renal cell tumours. J Pathol, 1997. 183(2): p.
  • PRCC is histologically characterized by the presence of f ⁇ brovascular cores with tumor cells arranged in a papillary or tubopapillary configuration.
  • the majority of PRCC tumors show indolent behavior, and have a limited risk of progression and mortality, but a distinct subset displays highly aggressive behavior.
  • Amin, M.B., CL. Corless, A.A. Renshaw, SfK . ⁇ ; tickoo, JV Rubus, and' D.S. Schultz, Papillary (chromophil) renal cell carcinoma: histomorphologic characteristics and evaluation of conventional pathologic prognostic parameters in 62 cases. Am J Surg Pathol, 1997. 21(6): p. 621-35.
  • Type 2 is characterized by the presence of large tumor cells with eosinophilic cytoplasm and pseudostratification. Generally, Type 2 tumors have a poorer prognosis than Type 1 tumors. Mejean, A., V. Hopirtean, J.P. Bazin, F. Larousserie, H. Benoit, Y. Chretien, N. Thiounn, and B. Dufour, Prognostic factors for the survival of patients with papillary renal cell carcinoma: meaning of histological typing and multifocality. J Urol, 2003. 170(3): p. 764-7.
  • Type 2 tumors were assessed as being of low Fuhrman nuclear grade despite pleomorphic nuclei being defined as a characteristic of Type 2 tumors. More recently, Allory et al (Allory, Y., D. Ouazana, E. Boucher, N. Thiounn, and A. Vieillefond, Papillary renal cell carcinoma. Prognostic value of morphological subtypes in a clinicopathologic study of 43 cases. Virchows Arch, 2003. 442(4): p. 336-42) classified only 1/13 (8%) as low-grade Type 2 tumors using a modified criterion.
  • PRCC can be effectively distinguished from the other major subtypes of RCC using gene classifiers, from which ⁇ -methylacyl-CoA racemase (AMACR) has been additionally validated as a useful immunohistochemical marker.
  • AMACR ⁇ -methylacyl-CoA racemase
  • Tretiakova, M.S., S. Sahoo, M. Takahashi, M. Turkyilmaz, NJ. Vogelzang, F. Lin, T. Krausz, B.T. Teh, and XJ. Yang Expression of alpha-methylacyl-CoA racemase in papillary renal cell carcinoma. Am J Surg Pathol, 2004. 28(1): p. 6976.
  • no distinct molecular subclasses of PRCC have been identified using this marker.
  • 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.
  • 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
  • Keratins are proteins that compose the 8-nm intermediate filaments in epithelial cells.
  • CK7 or KRT7, is a type II keratin of simple nonkeratinizing epithelia. It is expressed in multiple organs, with substantial expression previously observed in lung, bladder, mesothelium, hair follicle, and ductal structures.
  • DNA topoisomerases are enzymes that control and alter the topologic states of
  • Topoisomerase II from eukaryotic cells catalyzes the relaxation of supercoiled DNA molecules, catenation, decatenation, knotting, and unknotting of circular DNA. It appears likely that the reaction catalyzed by topoisomerase II involves the crossing-over of 2 DNA segments.
  • the inventors Using a clinically well-characterized patient population, the inventors correlated the global gene expression profiling of PRCC 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 two highly distinct molecular PRCC subclasses with morphological correlation, thus enabling the inventors to identify specific molecular signatures for each subclass of PRCC tumors.
  • the discovery of a set of differentially expressed genes for each subclass provides a basis for explaining differences in tumor aggressiveness and clinical outcome.
  • the methods and compositions described herein permit identification of proteins whose detection provide an early diagnostic approach to PRCC proteins as well as drug targets for the products of these genes.
  • a particular gene is differentially regulated in one subclass of PRCC, 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 invention provides a nucleic acid probe or set of probes (preferably between two and 139 in number) and a microarray comprising these markers as probes for the gene expression levels that are characteristic of PRCC tumor tissue.
  • a nucleic acid probe or set of probes preferably between two and 139 in number
  • a microarray comprising these markers as probes for the gene expression levels that are characteristic of PRCC 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 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 PRCC.
  • the probes are preferably any of SEQ ID NOS.: 1-139 inclusive.
  • the nucleic acid sequences hybridize to the probes under high stringency conditions.
  • the microarray may comprise at least about ten probes, more preferably one hundred probes, which probes are complementary to nucleic acid sequences expressed differentially in aggressive as compared to non-aggressive types of PRCC. 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
  • PRCC in a renal tumor tissue sample the relative expression of genes in a subject's PRCC 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.1-139.
  • Another method discovered by the inventors includes evaluating the aggressiveness of PRCC in a patient by detecting the level of expression in a renal tumor tissue sample of two or more genes from Table 4, wherein differential expression of the genes in Table 4 indicates whether the PRCC is aggressive or non-aggressive.
  • the genes in Table 6 are evaluated for aggressiveness of PRCC in a patient.
  • 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.
  • fluorescent labels hybridization is detected as a fluorescent signal bound to the probe.
  • Figures IA- ID show images of four categories of PRCC tumors fixed in buffered formalin and hexatoxylin-eosin stain (Type 1, Type 2A, Typel/2A mixed, Type 2B, respectively).
  • Figure IE plots expression profiles by first two principal components grouped into
  • Type 1 (thick ring symbol), Type 2 (solid) and mixed Type I/Type 2 (thin ring symbol) tumors.
  • Figure IF plots expression profiles by first two principal components grouped into Class 1 (solid) and Class 2 (ring) tumors.
  • Figure IG shows survival curves of Type 1, Type 2, and mixed Type I/Type 2 tumors, with Type 1 and mixed Type I/Type 2 curves overlapping.
  • Figure IH shows survival curves of Class 1 and Class 2 tumors.
  • Figure 2A is a heatmap showing hierarchical clustering of thirty-four PRCC tumor samples, showing distinct clustering of Class 1 (right) and Class 2 (left) tumors.
  • Figure 2B shows a comparative genomic microarray analysis inferred from cytogenic profiles of thirty- four PRCC tumor samples.
  • Figures 3A-L show images of formalin-fixed paraffin-embedded PRCC samples by H&E, CK7, and TopII ⁇ immunostaining in Type 1 (3A-C), Type 2A (3D-F), and Type
  • Figure 4A shows a prediction analysis of microarrays, depicting cross-validated misclassification error over a range of shrinking gene thresholds.
  • Figure 4B shows cross-validated predictions of tumor Class 1 (left) and Class 2
  • 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, copies thereof, or their analogues as described below. They may be poly- or oligonucleotides, without limitation of length.
  • 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 (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • 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.
  • 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 PRCC.
  • Dysregulation of Gl /S and G2/M checkpoint genes were found in Class 1 and Class 2 tumors respectively, alongside characteristic chromosomal aberrations.
  • the inventors also identified a seven-transcript predictor that classified samples on cross-validation with 97% accuracy. Immunohistochemistry confirmed high expression of cytokeratin 7 in Class 1 tumors, and of topoisomerase Il ⁇ in Class 2 tumors.
  • Class 2 PRCC may be distinguished from Class 1 (Type 1/ mixed Type 1 and 2 A / Type 2 A tumors) by the following characteristics: larger gross tumor size, higher nuclear grade (three to four), decreased CK7 staining and increased TopII ⁇ staining, higher rate of metastases at surgery and poorer patient survival. Morphologic findings of less specificity include larger cell size and eosinophilic cytoplasm in Class 2 tumors.
  • 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 al, Nature (2000) 405 (6788):827-836).
  • the underlying concept of the microarray depends on base-pairing (hybridization) between purine and pyrimidine bases following the rules of Watson-Crick base pairing.
  • DNA microarrays (DNA "chips") 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 al, (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 hydroxy 1-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 fiuorescently tagged nucleic acid sample into a chamber to hybridize with complementary oligonucleotides on the microarray slide; 1 aser 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 I a cDNA probe (500-5,000 bases) is immobilized to a solid surface such as glass using robotic spotting and exposed to a set of targets either separately or in a mixture. This method, traditionally called "DNA microarray,” is considered to have been developed at Stanford University (Ekins, R et al, Trends in Biotech (1999) 17:217-218).
  • 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), or peptide-nucleic acids (PNA).
  • probes that are "natural" oligo- or polynucleotides (oligomers of 20 ⁇ 80 bases), oligonucleotide analogues e.g., with phosphorothioate, methylphosphonate, phosphoramidate, or 3'-aminopropyl backbones), or peptide-nucleic acids (PNA).
  • 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 GIBCO-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 PRCC 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 an 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-2-
  • 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, ⁇ -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, coumarin 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.
  • PRCC which is the focus of the present invention
  • a database of known patient genetic profiles can be used to categorize each new PRCC patient.
  • the gene expression profile of the newly diagnosed PRCC patient is compared to the known PRCC molecular database of patients, such as that described herein based on thirty-four 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. If the newly diagnosed patient's gene expression profile most closely resembles the profile of aggressive PRCC, that patient will be so classified and treated accordingly, i.e., with more aggressive measures. Correspondingly, if 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 PRCC, particularly those that are associated with its aggressive form. A number of approaches are available.
  • PRCC 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.
  • PRCC 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.
  • HTS high-throughput screening
  • the molecular alterations in the cell line cells can be measured at the mRNA level
  • EIA enzyme immunoassays
  • RIA radioimmunoassay
  • flow cytometry 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).
  • the protein products of genes that are upregulated in PRCC are targets for ealy diagnostic assays of PRCC 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 3 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 H, 12 5 ⁇ I, 131 ⁇ I, 3S £ S, and
  • 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 tetramethylrhodamines, 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).
  • 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.
  • 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,
  • 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, 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 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 probes that are complementary to and thereby detect cells in which a gene associated with PRCC 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 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. Sci. 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. Sci. 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. Sci. 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. Sci. 83:4794-4798 (1986); Izant J.G. et al., Cell 36:1007-1015 (1984); Izant, J.G. et al., Science 229:345- 352 (1985); De Benedetti, A. et al., Proc. Natl. Acad. Sci.
  • the antisense oligonucleotides may range from 6 to 50 nucleotides, and may be as large as 100 or 200 nucleotides.
  • 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.
  • 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), lentiviruses, 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, R.J., 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,
  • 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 ai, Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081).
  • expression level can be detected by quantitative reverse transcription PCR if the the sample DNA is in formalin-fixed paraffin-embedded tissue.
  • Example 1- Patients and tumor samples
  • Tumor tissue was flash-frozen in liquid nitrogen immediately after nephrectomy and stored at -80 degrees Celsius. Portions of the tumors were fixed in buffered formalin and hematoxylin-eosin stained slide for all cases were centrally reviewed for the diagnosis of PRCC, except for one case (P 30), where slides were not available and histological description from the pathology report was used for subclassification. All 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.
  • Figure IA shows Type 1 PRCC (Class 1) with basophilic cytoplasm (Furhman grade 2).
  • Figure IB shows Type 2A PRCC (Class 1) with eosinophilic cytoplasm (Furhman grade 2).
  • Figure 1C shows mixed Type 1 and 2A PRCC (Class 1) with combined Type 1 (left) and Type 2 A (right) components (Furhman grade 2).
  • Figure ID shows Type 2B PRCC (Class 2) with eosinophilic cytoplasm and pseudostratified layers of tumor cells (Furhman grade 4).
  • DOD died of disease
  • DOO died of other causes
  • AWC alive with cancer
  • NED no evidence of disease.
  • RNA was used to prepare antisense biotinylated RNA.
  • a subset of cases was spiked with external poly- A RNA positive controls (Affymetrix, CA).
  • Synthesis of single-stranded and double- stranded complementary DNA was performed with the use of T7-oligo (dT) primer (Affymetrix).
  • In-vitro transcription was performed using Enzo Bioarray Transcript Labeling Kit (Enzo, NY). The biotinylated cRNA was subsequently fragmented, and 120ug was hybridized to each array at forty-five degrees Celsius over sixteen hours.
  • the HGU133 Plus 2.0 GeneChips contain 54,675 probe sets, representing approximately 47,000 transcripts and variants. Scanning was performed in a GeneChip 3000 scanner. Quality assessment of performed in GeneChip Operating System (GCOS) 1.1.1 (Affymetrix) using global scaling to a target signal of 500. Quality assessment was performed using denaturing gel electrophoresis. The manufacturer's recommended protocol (GeneChip Expression Analysis Technical Manual, Affymetrix, April 2003) was followed for expression profiling. Median background was seventy-three, median scaling factor was 3.06, and median GADPH 375' ratio was 1.03, indicative of a high overall array and RNA quality. All data will be uploaded to the Gene Expression Omnibus under a pending accession number.
  • GCOS GeneChip Operating System
  • the inventors analyzed the profiles based on this morphologic subtyping into two classes (Class 1 corresponding to Type 1, low-grade Type 2 and mixed Type 1 and low-grade Type 2 tumors, and Class 2 corresponded to high-grade Type 2 tumors) from a molecular viewpoint. Visualization of principal components then demonstrated distinct differential between expression profiles of Class 1 and Class 2 tumors, consistent with distinct tumor subclasses ( Figure IF).
  • *D(i) is a modified t-statistic calculated by SAM.
  • FIG. 2A A hierarchical clustering of the tumor samples based on the one hundred up- expressed transcripts are shown in Figure 2A.
  • Hierarchical clustering of tumor samples by the top one hundred differentially expressed genes in each PRCC class is shown in Figure 2A.
  • rows represent individual oligonucleotide probes and columns represent individual tumor samples.
  • Complete linkage clustering and a Euclidean distance metric was used, and values were scaled by row.
  • the left cluster represents Class 2 tumors, corresponding to all Type 2B papillary tumors.
  • the right cluster represents Class 1 tumors corresponding to all Type 1 and Type 2A tumors.
  • class scores are linear discriminant scores for each class as described in Tusher, et al. Only the tumor of P 30, initially reported as a Type 2 tumor with grade 2, which the inventors were unable to confirm histologically, persistently classified as a Class 2 tumor, rather than as a Class 1 tumor, throughout these multiple shrinkage thresholds.
  • Aneuploidy is well established as a key driver of global gene expression, and regional DNA copy number correlates well with regional expression in cancer (Hughes, T.R., CJ. Roberts, H. Dai, A.R. Jones, M.R. Meyer, D. Slade, J. Burchard, S. Dow, T.R. Ward, M.J. Kidd, S.H. Friend, and M.J. Marton, Widespread aneuploidy revealed by DNA microarray expression profiling. Nat Genet, 2000. 25(3): p. 333-7), which also has been demonstrated in RCC classification. Furge, K.A., K.A. Lucas, M. Takahashi, J. Sugimura, E.J.
  • PRCC typically shows frequent trisomy 7, 12, 16, 17 and 20 (Amin, M.B., CL. Corless, A.A. Renshaw, S.K. Tickoo, J. Kubus, and D. S. Schultz, Papillary (chromophil) renal cell carcinoma: histomorphologic characteristics and evaluation of conventional pathologic prognostic parameters in 62 cases. Am J Surg Pathol, 1997. 21(6): p.
  • Relative expression profiles R were generated from the single channel tumor expression profiles (T) and the mean expression values of the twelve single channel kidney cortical expression profiles (N) such that R+log 2 (T) - log 2 (N).
  • CGMA ( Figure 2B). CGMA profiles of PRCC were generated from tumor: kidney cortical tissue expression ratios. As shown in Figure 2B, comparative genomic microarray analysis shows inferred cytogenetic profiles of the thirty-four tumor samples. 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. Group 1 tumors correspond to samples above the white bar, and Group 2 tumors correspond to samples above the black bar.
  • the present results support a report that loss of heterozygosity at 9q is associated with reduced survival. Renshaw, A.A. and CL. Corless, Papillary renal cell carcinoma. Histology and immunohistochemistry. Am J Surg Pathol, 1995. 19(7): p. 842-9.
  • Gl/S checkpoint regulation cyclin D2, cyclin-dependent kinase 6, retinoblastoma-like 2 and p21Cipl
  • genes involved in G2/M checkpoint regulation cyclin Bl, cyclin B2 and topoisomerase II alpha
  • the present invention highlights dysregulation of Gl/S checkpoint genes in Class 1 PRCC and dysregulation of G2/M checkpoint genes in Class 2 PRCC, as the most highly ranked pathways identified in the differentially expressed genes. Details of individual gene expression in the 139 transcripts are shown in Table 6. Table 6
  • Relative fold change is expressed in terms of expression in Class 2 tumors relative to Class 1 tumors
  • oligonucleotide probe sets corresponding to c-met were identified as being upexpressed in Class 1 tumors, ranging between 2- to 3 -fold upexpression.
  • mutations of the c-met proto-oncogene have been implicated in hereditary Type 1 PRCC (Schmidt, L., K. Junker, G. Weirich, G. Glenn, P. Choyke, I. Lubensky, Z. Zhuang, M. Jeffers, G. Vande Woude, H. Neumann, M. Walther, W.M. Linehan, and B. Zbar, Two North American families with hereditary papillary renal carcinoma and identical novel mutations in the MET proto-oncogene. Cancer Res, 1998.
  • DNA topoisomerase II alpha (TopII ⁇ ) is a diagnostic marker for Class 2 tumors.
  • TopII ⁇ DNA topoisomerase II alpha
  • doxorubicin and etoposide could be used in a therapeutic trial of PRCC.
  • G2 arrest occurs in response to these agents (Clifford, B., M. Beljin, G.R. Stark, and W.R. Taylor, G2 arrest in response to topoisomerase II inhibitors: the role of p53.
  • CK7 immunohistochemistry To validate the gene predictor and to derive immunohistochemical markers, the inventors used immunohistochemistry to confirm high protein expression of CK7 in Class 1 tumors and of Topo Il ⁇ in Class 2 tumors. CK7 immunoreactivity has been previously reported to the vast majority of PRCC (Renshaw, A.A. and CL. Corless, Papillary renal cell carcinoma. Histology and immunohistochemistry. Am J Surg Pathol, 1995. 19(7): p. 842-9), but more recent studies suggested that CK may differentiate Type 1 and Type 2 tumors.
  • 6976 using mouse monoclonal antibodies specific for cytokeratin 7 (CK7, 1 :50 dilution, Dako, Carpinteria, CA) and DNA topoisomerase II alpha (TopII ⁇ , 1/20 dilution, Vector Laboratories, Burlingame, CA).
  • CK7 cytokeratin 7
  • Dako Dako
  • Carpinteria CA
  • TopII ⁇ DNA topoisomerase II alpha
  • PRCC samples which had undergone microarray analysis (ten Class 1 tumors, eight Class 2 tumors), as well as an independent set of fifteen tumors (ten Class 1 tumors, five Class 2 tumors).
  • the thirteen Class 2 tumors were all high-grade Type 2 tumors.
  • the CK7 immunoreactivity was graded as negative ( ⁇ 0.1% positive tumor cells), focally positive (0.1-10% positive tumor cells) or positive (>10% positive tumor cells).
  • the TopII ⁇ immunoreactivity was graded as negative ( ⁇ 0.1% positive tumor cells), focally positive (0.1-10% positive tumor cells) or positive (>10% positive tumor cells).
  • the Mann- Whitney test was used to evaluate significance of the differential staining.
  • TopII ⁇ immunoreactivity was detected in normal kidney tissue. There was no apparent difference between Type 1 and low-grade Type 2 (Type 2A) tumors in CK7 and TopII ⁇ immunostaining. Summarizing the results, CK7 immunoreactivity was significantly higher in Class 1 tumors (p ⁇ 0.001), and TopII ⁇ immunoreactvity was significantly higher in Class 2 tumors (p ⁇ 0.001).
  • Figures 3A-C show Type 1 tumor stained with H&E (A), CK7 (B) and TopII ⁇
  • Figures 3D-F show low-grade Type 2 (Type 2A) tumor stained with H&E (D), CK7 (E) and TopII ⁇ (F).
  • Figures 3G-I show high-grade Type 2 (Type 2B) tumor, which was subjected to microarray analysis, stained with H&E (G), CK7 (H) and TopII ⁇ (I).
  • a renal tubule (arrow, H) stains positive for CK7 as an internal positive control, while all tumor cells are negative.
  • Figures 3J-L show high-grade Type 2 (Type 2B) tumor, which was not subjected microarray analysis, stained with H&E (J), CK7 (K) and TopII ⁇ (L).
  • a renal tubule (arrow, K) is positive for CK7, while all tumor cells are negative.

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Abstract

L'invention a pour objet une sonde d'acide nucléique ou un nouvel ensemble comprenant lesdites sondes dans un microréseau. La sonde ou l'ensemble de sondes sont utiles dans le pronostic de patients atteints par le carcinome cellulaire rénal papillaire (PRCC), où les types de la tumeur PRCC agressive et non agressive sont caractérisés par des profils d'expression différentielle des gènes qui s'hybrident avec au moins une desdites sondes. L'invention se rapporte également à des microréseaux et à des kits permettant de réaliser le profilage d'expression du tissu tumoral et à des procédés utilisant lesdits microréseaux et kits.
PCT/US2005/013260 2005-04-18 2005-04-18 Profilage d'expression genique par microreseau dans des classes de carcinome cellulaire renal papillaire WO2006112842A2 (fr)

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