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US20130203619A1 - Methods for Detecting, Diagnosing and Treating Human Renal Cell Carcinoma - Google Patents

Methods for Detecting, Diagnosing and Treating Human Renal Cell Carcinoma Download PDF

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US20130203619A1
US20130203619A1 US13/718,871 US201213718871A US2013203619A1 US 20130203619 A1 US20130203619 A1 US 20130203619A1 US 201213718871 A US201213718871 A US 201213718871A US 2013203619 A1 US2013203619 A1 US 2013203619A1
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renal cell
cell carcinoma
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John Alton Copland, III
Bruce A. Luxon
Christopher G. Wood
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University of Texas System
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates generally to the field of cancer research. More specifically, the present invention relates to gene expression profiling for human renal cell carcinoma.
  • Renal cell carcinoma represents a major health issue.
  • the American Cancer Society predicts 31,900 new cases will be diagnosed in the United States alone in the year 2003, with 11,900 people dying of the disease.
  • renal cell carcinoma can be surgically resected for cure using a variety of approaches.
  • metastatic progression however, renal cell carcinoma is incurable, and existing systemic therapies are largely ineffective in impacting disease response or patient survival.
  • the lack of effective systemic therapy for metastatic renal cell carcinoma is, in part, due to a fundamental lack of understanding of the molecular events that result in cellular transformation, carcinogenesis, and progression in human kidney.
  • novel renal cell carcinoma markers to be used for detection, diagnosis and development of effective therapy against the disease remains a high priority.
  • the prior art is deficient in understanding the molecular differences between renal cell carcinoma and normal renal epithelium.
  • the present invention fulfills this need in the art by providing gene expression profiling for these two types of tissues.
  • the present invention identifies genes with a differential pattern of expression between different subtypes of renal cell carcinomas (RCC) and normal renal epithelium. These genes and their products can be used to develop novel diagnostic and therapeutic markers for the treatment of renal cell carcinomas.
  • RCC renal cell carcinomas
  • Genomic profiling of conventional renal cell carcinoma tissues and patient-matched normal kidney tissue samples was carried out using stringent statistical analyses (ANOVA with full Bonferroni corrections).
  • Subtypes of renal cell carcinoma include stage I, II, III, and IV (reflecting differences in tumor size, lymph node and organ metastasis), stage I papillary renal cell carcinoma, and benign oncocytoma.
  • Hierarchical clustering of the expression data readily distinguished normal tissue from renal cell carcinomas. The identified genes were verified by real-time FCR and immunohistochemical analyses.
  • Different subtypes of conventional renal cell carcinomas can be diagnosed either by drawing blood and identifying secreted gene products specific to renal cell carcinoma or by doing a biopsy of the tissue and then identify specific genes that are altered when renal cell carcinoma is present.
  • An example of when this may be especially important is in distinguishing the deadly conventional renal cell carcinomas (account for 85% of all renal cell carcinomas) from renal oncocytoma (benign renal cell carcinoma) as well as identifying the histologic subtypes of papillary and sarcomatoid renal cell carcinoma. Identification of specific genes will also help in determining which patients will have a good prognosis verses that of a poor prognosis.
  • subsets of genes identified in the present invention can be developed as targets for therapies that could cure, prevent, or stabilize the disease. Thus, results from the present invention could be used for diagnosis, prognosis, and development of therapies to treat or prevent renal cell carcinoma.
  • conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
  • conventional or clear cell renal cell carcinoma is detected based on decreased expression of type III TGF- ⁇ receptor.
  • stage I conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
  • the present invention also provides methods of detecting stage II conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
  • the present invention also provides methods of detecting papillary renal cell carcinoma or benign oncocytoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
  • a method of targeting conventional or clear cell renal cell carcinoma cells based on generating antibodies or small molecules directed against a cell surface molecule over-expressed in conventional renal cell carcinoma cells.
  • FIG. 1A shows hierarchical clustering of genes expressed in normal renal cortex (12 patient tissue samples) verse stage I conventional renal cell carcinoma (6 patient tissue samples). Red indicates that a gene is highly expressed and green is indicative of low expression.
  • FIG. 1B shows hierarchical clustering of genes expressed in normal renal cortex (12 patient tissue samples) verse stage II conventional renal cell carcinoma (6 patient tissue samples). Red indicates that a gene is highly expressed and green is indicative of low expression.
  • FIG. 1C shows hierarchical clustering of genes selected from a Venn analysis in which the chosen genes were expressed in common in both stage I and II at a 99% confidence level. One hundred eighty eight genes were depicted in FIG. 1C .
  • C cancer cells
  • N normal cells
  • FIG. 2 shows TGF- ⁇ 1 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR TGF- ⁇ 1 mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
  • FIG. 3 shows TGF- ⁇ mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF- ⁇ mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
  • FIG. 4 shows adrenomedulin mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. Adrenomedulin mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
  • FIG. 5 shows TGF-t ⁇ 2 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF- ⁇ 32 mRNA levels were not altered between normal and tumor matched samples.
  • FIG. 6 shows TGF-133 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF- ⁇ 3 mRNA levels were not altered between normal and tumor matched samples.
  • FIG. 7 shows tumor suppressor gene Wilms Tumor 1 (WT1) mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. WT1 mRNA levels were down-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
  • FIG. 8 shows von Hippel Lindau mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. A small percentage of tumor tissues demonstrated attenuated von Hippel Lindau mRNA levels when compared to matched normal tissue
  • FIG. 9 shows calbindin mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. Calbindin mRNA was completely lost in all stage I renal cell carcinoma. p ⁇ 0.05 compared to matched control. *Stage I tumor: 0 ⁇ 0; stage III tumor: 0.0009 ⁇ 0.0004; stage IV tumor: 0.003 ⁇ 0.0004/
  • FIG. 10 shows MUC1 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. MUC1 mRNA levels were down-regulated in all tumor tissues as early as stage I. *p ⁇ 0.05 compared to matched control.
  • FIGS. 11A-11B show stepwise loss of type III ⁇ receptor (TBR3) and type II TGF- ⁇ receptor (TBR2) mRNA expression during renal cell carcinogenesis and progression in patient tissue samples.
  • FIG. 11A shows gene array data from 10 patients—five diagnosed with localized renal cell carcinoma and five with metastatic disease. ‘+’ (P ⁇ 0.05) indicates statistical difference for TBR3 mRNA levels as compared to normal tissue and ‘*’ (P ⁇ 0.28) indicates statistical difference for TBR2 mRNA levels as compared to normal controls. Data are expressed as mean ⁇ s.e.
  • FIG. 11B shows real-time RT-PCR verification of TBR1, TBR2, and TBR3 mRNA levels of tissue samples described in FIG. 11A . Data are expressed as mean ⁇ s.d.
  • FIG. 12 shows immunohistochemistry of patient tissue demonstrating loss of type III ⁇ receptor (TBR3) expression (top row) in all tumors, loss of type II ⁇ receptor (TBR2) expression (middle row) in patients diagnosed with metastatic tumors, and no change in type I ⁇ receptor (TBR1) protein expression (bottom row).
  • TBR3 loss of type III ⁇ receptor
  • TBR2 loss of type II ⁇ receptor
  • TBR1 type I ⁇ receptor
  • FIG. 13 demonstrates down-regulation of TGF-3-regulated genes in human tumor tissues by real-time PCR. Genes known to be up-regulated by ⁇ are suppressed in tumor tissues. Down-regulation of collagen IV type 6, fibulin 5, and connective tissue growth factor (CTGF) mRNA in tumor tissues were compared to matched normal tissue controls. Values were normalized to 18 s mRNA. Each matching tumor value was compared to its respective normal control. The mean ⁇ s.d. was calculated for each sample group with n values of 10-15 matched samples.
  • CTGF connective tissue growth factor
  • FIGS. 14A-14B show tumor cell lines that lose type III ⁇ receptor (TBR3) and type I TGF- ⁇ receptor (TBR2) expression.
  • FIG. 14A shows semi-quantitative RT-PCR measurements of mRNA levels of TBR1, TBR2, and TBR3 for UMRC3, UMRC6 and normal renal epithelial (NRE) cells.
  • FIG. 14B shows immunohistochemistry of protein expression for TBR1, TBR2, and TBR3 ( ⁇ 40 magnification).
  • FIGS. 15A-15B show loss of type III TGF- ⁇ receptor (TBR3) and type II ⁇ receptor (TBR2) expression in renal tumor cell lines correlate with loss of TGF-3-regulated growth inhibitory and transcriptional responses.
  • FIG. 15A shows cell proliferation was inhibited as assessed by DNA content 3 days after ⁇ treatment. Percent of each respective untreated control was used for comparisons.
  • Transient transfection using 3TP/lx along with a renilla luciferase control demonstrates loss of responsiveness to 2 ng/ml TGF- ⁇ 1 with loss of TGF- ⁇ receptor expression ( FIG. 15B ).
  • Firefly luciferase activity was normalized using the ratio of firefly luciferase/renilla luciferase. Data are expressed as mean ⁇ s.d.
  • FIG. 16A demonstrates RT-PCR derived mRNA expression of type III ⁇ receptor (TBR3), type II ⁇ receptor (TBR2), and type I ⁇ receptor (TBR1) in UMRC3 cells and cells stably transfected with TBR2 and TBR3.
  • FIG. 16B shows UMRC3 cells stably transfected with type II TGF- ⁇ receptor (UMRC3+TBR2) or type II and type III TGF- ⁇ receptor (UMRC3+TBR2+TBR3) demonstrated attenuated cell proliferation following the administration of exogenous TGF- ⁇ 1 as compared to that of UMRC3 cells.
  • FIG. 16C shows UMRC3 cells, UMRC3+TBR2 cells, and UMRC3+TBR2+TBR3 stable cell lines transfected with 3TP/lux were treated with or without TGF- ⁇ and examined for luciferase activity.
  • FIG. 16D shows real-time PCR measuring mRNA levels for collagen IV type 6 in UMRC3, UMRC3+TBR2 cells, and UMRC3+TBR2+TBR3 cells in the presence of 2 ng/ml TGF- ⁇ 1 for 24 h.
  • FIG. 16E shows colony formation assay demonstrates that UMRC3+TBR2+TBR3 cells have completely lost anchorage-independent growth, while attenuated growth in UMRC3+TBR2 cells occurs as compared to that of UMRC3 cells. The number of colonies were stained and counted after 45 days of growth. Data are expressed as mean ⁇ s.d.
  • FIG. 17A shows growth inhibition after re-expressing human type III TGF- ⁇ 3 receptor (TBR3) in UMRC3 cells.
  • UMRC3 cells were stably transfected with TBR3 or infected using an adenoviral vector expressing TBR3.
  • Cells were plated in culture dishes at 20,000 cells/well. Cell number was determined at the indicated times using a Coulter cell counter.
  • FIG. 17B shows RT-PCR data demonstrating the mRNA expression levels of type I, II, or III TGF- ⁇ receptors (TBR1, TBR2, TBR3) in UMRC3 cells in the presence or absence of the adenoviral vector expressing TBR3. Unmodified UMRC3 cells only express TBR1.
  • FIG. 18 shows re-expression of human type II or III TGF- ⁇ receptors (TBR2 or TBR3) inhibits tumor growth in nude mice.
  • TBR2 or TBR3 TGF- ⁇ receptors TBR2 or TBR3
  • FIG. 19 shows hierarchical clustering of genes expressed in normal renal cortex verse stage I papillary renal cell carcinoma. Red indicates that a gene is highly expressed and green is indicative of low expression.
  • FIG. 20 shows hierarchical clustering of genes expressed in normal renal cortex verse benign oncocytoma. Red indicates that a gene is highly expressed and green is indicative of low expression.
  • FIG. 21 shows venn analysis of gene distribution among stage I renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma.
  • FIG. 22 shows venn analysis of gene distribution among stage II renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma.
  • High-throughput technologies for assaying gene expression such as high-density oligonucleotide and cDNA microarrays, offer the potential to identify clinically relevant genes differentially expressed between normal and tumor cells.
  • the present invention discloses a genome-wide examination of differential gene expression between renal cell carcinomas (RCC) and normal renal epithelial cells.
  • the present invention detects genes that have differential expression between renal cell carcinomas and normal renal epithelial cells. The known function of some of these genes may provide insight into the biology of renal cell carcinomas while others may prove to be useful as diagnostic or therapeutic markers against renal cell carcinomas.
  • Subtypes of renal cell carcinomas disclosed herein include stage I, II, III, and IV renal cell carcinomas (reflecting differences in tumor size, lymph node and organ metastasis), stage I papillary renal cell carcinoma, and benign oncocytoma.
  • the present invention provides methods of detecting conventional renal cell carcinoma based on determining the expression level of a number of genes that are found to have 2-fold or higher differential expression levels between tumor and normal tissue.
  • biological samples e.g. tissue samples, serum samples, urine samples, saliva samples, blood samples or biopsy samples
  • tissue samples serum samples, urine samples, saliva samples, blood samples or biopsy samples
  • gene expression at RNA or protein level is compared to that in normal tissue.
  • the normal tissue samples can be obtained from the same individual who is to be tested for renal cell carcinoma.
  • gene expression can be determined by DNA microarray and hierarchical cluster analysis, real-time PCR, RT-PCR, or northern analysis, whereas secreted gene products can be measured in blood samples by standard procedures.
  • a method of detecting conventional or clear cell renal cell carcinoma based on differential expression of one or more of the following genes or proteins: TGF- ⁇ 1, TGF- ⁇ , adrenomedulin, fibroblast growth factor 2 (FGF2), vascular epidermal growth factor (VEGF), osteonectin, follistatin like-3, inhibin beta A, spondin 2, chemokine X cytokine receptor 4 (CXCR4), fibronectin, neuropilin 1, frizzled homolog 1, insulin-like growth factor binding protein 3, laminin alpha 3, integrin beta 2, semaphorins 6A, semaphorins 5B, semaphorins 3B, caspase 1, sprouty 1, CDH16, PCDH9, compliment component 1-beta, compliment component 1-alpha, compliment component 1-gamma, CD53, CDW52, CD163, CD14, CD3Z, CD24, RAP1, angiopoietin 2, cytokine knot secreted
  • a method of detecting conventional renal cell carcinoma by examining the expression level of type III TGF- ⁇ receptor, wherein decreased expression of type III TGF-b receptor indicates the presence of renal cell carcinoma.
  • the expression level of type III TGF- ⁇ receptor can be determined at the mRNA or protein level.
  • the present invention also provides methods of detecting stage I conventional renal cell carcinoma, stage II conventional renal cell carcinoma, stage I papillary renal cell carcinoma, or benign oncocytoma based on over-expression or down-regulation of a number of genes identified in the present invention.
  • the present invention discloses a number of genes that are up- or down-regulated specifically in these subtypes of renal cell carcinoma. Determining the expression levels of these genes would provide specific diagnosis for these different subtypes of renal cell carcinoma.
  • stage I conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 1, (ii) down-regulation of one or more genes listed in Table 2, or (iii) over-expression of one or more genes listed in Table 1 and down-regulation of one or more genes listed in Table 2.
  • stage II conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 3, (ii) down-regulation of one or more genes listed in Table 4, or (iii) over-expression of one or more genes listed in Table 3 and down-regulation of one or more genes listed in Table 4.
  • stage I or stage II conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 5, or (ii) down-regulation of one or more genes listed in Table 6.
  • stage I papillary renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 8, (ii) down-regulation of one or more genes listed in Table 9, or (iii) over-expression of one or more genes listed in Table 8 and down-regulation of one or more genes listed in Table 9.
  • benign oncocytoma can be detected based on (i) over-expression of one or more genes listed in Table 10, (ii) down-regulation of one or more genes listed in Table 11, or (iii) over-expression of one or more genes listed in Table 10 and down-regulation of one or more genes listed in Table 11.
  • a number of genes that are up-regulated in stage I renal cell carcinoma (RCC), stage II RCC tumor, stage I papillary RCC, and benign oncocytoma can be linked with a therapeutic drug to deliver drug to the tumor tissue, or be linked with dye, nanoparticle or other imaging agents for cancer imaging.
  • novel genes identified herein for the first time include, but are not limited to, the following genes: calcitonin receptor-like (206331_at; 210815_s_at); receptor (calcitonin) activity modifying protein 2 (RAMP2; 205779_at); endothelin receptor type B (206701_x_at); beta 2 integrin (202803_s_at); alpha 5 integrin (201389_at); chemokine X cytokine receptor 4 (CXCR4); fibronectin; neuropilin 1 (212298_at; 210510_s_at); CD24; CD14; Cd163; CD53; Compliment Componenet 1-beta, 1-alpha, and 1-gamma; CDH4; integrin beta2; ADAM28; FK506 binding protein; collagen Valpha2; tumor necrosis factor receptor superfamily, member 6; tumor necrosis factor receptor superfamily, member 5; tumor necrosis factor (ligand) superfamily, member 13b; tumor necrosis
  • a method of treating conventional or clear cell renal cell carcinoma involves replacing tumor suppressor genes (e.g., via gene therapy) whose expression is down-regulated in tumor tissues or introducing a molecule that induces the down-regulated gene to be re-expressed in the tumor.
  • the present invention discloses a number of genes that are down-regulated in stage I renal cell carcinoma (RCC), stage II RCC tumor, stage I papillary RCC, and benign oncocytoma.
  • genes identified in stage I and/or II RCC tumors include, but are not limited to, CDKN1, secreted frizzled related protein 1, semaphoring 6D, semaphoring 3B, CDH16, TNF alpha, calbindin D28, defensin beta1, beta-catenin interacting protein 1, GAS1, vitamin D receptor, Kruppel-like factor 15. This method of treatment can be combined with other therapies to provide combinatorial therapy.
  • stage I and stage II renal cell carcinoma would also be useful for determining patient prognosis.
  • genes or gene products i.e., proteins
  • these genes or gene products would have the unique characteristic of being altered in tumor verses normal samples in a subset of patients.
  • basic transcription element binding protein 1 is down-regulated in 7 out of 12 renal cell carcinoma tumors.
  • CD164 decreased 5/12; Map kinase kinase kinase 7, increased 6/12; Endoglin, increased 7/12; SERPIN A1, increased 6/12; Metalloprotease 11 (MMP11), increased 7/12; Integrin 3 alpha, increased 4/12; carbonic anhydrase II, decreased 7/12; protein tyrosine kinase 2, increased 4/12; fibroblast growth factor 11, increased 6/12; fibroblast growth factor 2, increased 7/12; VEGF B, increased 5/12.
  • MMP11 Metalloprotease 11
  • the levels of change may be a useful determinant of patient outcome and/or rationale for strategy of treatment course.
  • An example of this is found for chemokine (C-X-C motif) ligand 14 (CXCL14, 222484_s_at).
  • CXCL14, 222484_s_at chemokine (C-X-C motif) ligand 14
  • CXCL14, 222484_s_at chemokine (C-X-C motif) ligand 14
  • Renal tissue normal and tumor was transported to a sterile hood on ice and under sterile conditions. Tissue was dissected under the direction of a pathologist. The tissue was frozen in liquid nitrogen for isolation of RNA, DNA, and protein or processed to establish primary cell cultures. The tissue was fixed in formalin for immunohistochemistry and in situ hybridization and RNAlater (Ambion) for RNA isolation.
  • Primary normal renal epithelial (NRE) cell cultures were established using standard collagenase/Dnase techniques to digest tissue and isolate single cells. NREs were easily isolated and grew well in culture for up to 10 passages. These cells were further analyzed for homogeneity with regard to epithelial population using appropriate immunohistochemical markers such as vimentin, cytokeratin, and megalin.
  • Gene expression profiling was performed using Affymetrix HU95A oligonucleotide gene arrays (>12,600 genes) or HG-U133 A&B GeneChip® oligonucleotide microarrays (33,000+ probe sets).
  • Total RNA Trizol®, Ambion
  • the investigators analyzed metastatic disease defined by lesions in lymph nodes, adrenal, or other organs.
  • Data were analyzed by a combination of two-dimensional ANOVA, Affymetrix MAS5.0®, and hierarchical cluster analysis using Spotfire®. Procedure that were used to identify altered expression of large sets of genes, as well as other issues concerning microarray analyses can be found in a recent review article by Copland et al. (2003).
  • Separate tubes (singleplex) for one-step RT-PCR was performed with 50 ng RNA for both target genes and endogenous controls using TaqMan® one-step RT-PCR master mix reagent kit (Applied Biosystems).
  • the cycling parameters for one-step RT-PCR were: reverse transcription 48° C. for 30 min, AmpliTaq® activation 95° C. for 10 min, denaturation 95° C. for 15 s, and annealing/extension 60° C. for 1 min (repeat 40 times) on ABI7000®.
  • Duplicate C T values were analyzed with Microsoft Excel® using the comparative C T (DDC T ) method as described by the manufacturer (Applied Biosystems).
  • the amount of target (2 ⁇ DDCT ) was obtained by normalizing to an endogenous reference (18smRNA) and relative to a calibrator (normal tissue).
  • TBR1 type I TGF- ⁇ receptor
  • TBR2 type II TGF- ⁇ receptor
  • TBR3 type III TGF- ⁇ receptor
  • patient-matched normal renal and tumor tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks.
  • Consecutive sections were cut 5 um thick, deparaffinized, hydrated, and immunostained using antibodies recognizing human TBR1, TBR2, and TBR3 (1:100; Santa Cruz Biotechnology).
  • Biotinylated secondary antibody (1:600; Santa Cruz Biotechnology) was detected using avidin-biotin-peroxidase detection according to the manufacturer's instructions (Vectastatin Elite ABC kit; Vector Lab). All slides were lightly counterstained with hematoxylin before dehydration and mounting.
  • cells were plated on glass coverslips in wells. Prior to the detection of TGF- ⁇ receptor expression as described above, cells were fixed onto the coverslips with 3% formalin.
  • a primary goal of microarray analysis is to discover hidden patterns of differential expression within a large data field.
  • Normal renal cortical and primary tumor tissue with no metastasis were collected from patients diagnosed with local disease.
  • Normal tissue, primary tumor, and metastatic tissue were also collected from patients diagnosed with metastatic disease.
  • Comparison of patient-matched normal and tumor tissue allowed for the discovery of changes in mRNA levels between normal and tumor tissue, as well as local and metastatic disease.
  • FIG. 1 Heatmaps with two-way dendograms depicting genes specifically altered in tumor tissue as compared to normal renal cortex are shown in FIG. 1 .
  • FIG. 1A shows hierarchical clustering of genes expressed in normal renal cortex verses stage I conventional renal cell carcinoma.
  • FIG. 1B shows hierarchical clustering of genes expressed in normal renal cortex verses stage II renal cell carcinoma.
  • FIG. 1C shows hierarchical clustering of genes selected from a Venn analysis in which the chosen genes were expressed in common in both stage I and II at a 99% confidence level.
  • TGF- ⁇ 1, TGF- ⁇ and adrenomedulin mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts ( FIGS. 2-4 ), whereas TGF- ⁇ 2 and TGF- ⁇ 3 mRNA levels were not altered between normal and tumor matched samples ( FIGS. 5-6 ).
  • Tumor suppressor gene Wilms Tumor 1 was down-regulated in all stages of renal cell carcinoma ( FIG. 7 ). A small percentage of tumor tissues demonstrated attenuated von Hippel Lindau mRNA levels when compared to matched normal tissue ( FIG. 8 ). Calbindin mRNA was completely lost ( FIG. 9 ) while MUC1 was greatly attenuated in stage I renal cell carcinoma ( FIG. 10 ).
  • the present analysis identifies 278 genes that were up-regulated in stage I renal cell carcinoma, whereas 380 genes were up-regulated in stage II renal cell carcinoma. Among these genes, 82 were up-regulated in both stages I and II renal cell carcinoma. One hundred fifty nine genes were down-regulated in stage I renal cell carcinoma, whereas 195 genes were down-regulated in stage II RCC. Among these genes, 82 were down-regulated in both stage I and II renal cell carcinoma.
  • TBR1 type I TGF- ⁇ receptor
  • TBR2 type II TGF- ⁇ receptor
  • TBR3 type III TGF- ⁇ receptor
  • TBR2 was significantly reduced in primary lesions with metastatic disease (P ⁇ 0.028 by ANOVA). TBR2 was even more reduced in metastatic lesions. TBR3 expression was high in normal epithelium, but was significantly reduced in each of the five primary tumors with nonmetastatic disease (black bars). TBR3 expression was also reduced in primary tumors with metastatic lesions and in metastatic lesions themselves.
  • TBR1, TBR2, and TBR3 expression were subsequently completed real-time PCR analysis of TBR1, TBR2, and TBR3 expression in 16 primary tumors without metastases (plus paired normal epithelium) and nine samples of primary tumors with metastatic disease, paired metastatic lesions, and paired normal tissue. The data were consistent with those shown for the samples analyzed in FIG. 11A . TBR3 expression was significantly reduced in all tumors; whereas TBR2 expression was reduced in only 1/16 primary tumors without metastatic lesions, but was reduced in primary tumors with metastatic lesions (8/9). These data show that loss of TBR3 is an early event in renal cell carcinoma, strongly suggesting that TBR3 plays a critical role in renal cell carcinoma carcinogenesis.
  • TBR3 mRNA expression was also correlated with TNM scores (T, histological score; N, lymph node number; M, number of organ metastases) from patient samples (data not shown). TBR3 mRNA expression was suppressed in the earliest stage, stage I, and was found to be suppressed in all tumor stages (I-IV). In addition, loss of TBR2 in the primary tumor is significantly associated with acquisition of the metastatic phenotype and clinically manifests as metastatic progression.
  • Type III TGF- ⁇ receptor TBR3 mRNA expression in all tumors was associated with failure to detect TBR3 protein by immunohistochemistry ( FIG. 12 ).
  • Type I TGF- ⁇ receptor (TBR2) protein was detected in localized tumor (primary, no mets), but was not detectable in primary tumors with metastatic disease or in corresponding metastatic lesions.
  • Type I TGF- ⁇ receptor (TBR1) protein was detected in normal tissue and in all tumor samples.
  • TGF- ⁇ receptor expression would manifest as an attenuation of TGF- ⁇ mediated signal transduction, and would significantly alter the expression of TGF- ⁇ regulated genes.
  • 13 known TGF-3/Smad-regulated genes were down-regulated in renal cell carcinoma (Table 7).
  • the investigators verified loss of expression of three of these genes by comparing matched normal and tumor tissue.
  • Real-time PCR was used to measure the expression of Collagen IV type 6, fibulin-5, and connective-tissue growth factor (CTGF).
  • Collagen IV type 6 (gray bars) is an extracellular matrix protein that plays a critical role in the regulation of membrane integrity and cell signaling.
  • Fibulin-5 is a recently discovered TGF-3-regulated gene, which has tumor suppressor activity. Fibulin-5 is an extracellular matrix protein that is believed to signal through interaction with integrins. CTGF is a secreted protein involved in angiogenesis, skeletogenesis, and wound healing. CTGF enhances TGF- ⁇ 1 binding to TBR2, and CTGF and TGF- ⁇ collaborate to regulate the expression of extracellular matrix proteins during renal fibrosis. As summarized graphically in FIG. 13 , all the evaluated TGF- ⁇ -regulated genes were down-regulated in early tumor stages, suggesting that renal cell carcinoma undergoes loss of TGF- ⁇ responsiveness at an early stage.
  • TGF- ⁇ sensitivity is due, primarily, to loss of type III TGF- ⁇ receptor (TBR3) in early tumor development and further loss of sensitivity in metastatic disease is mediated through subsequent loss of type II TGF- ⁇ receptor (TBR2).
  • TBR3 type III TGF- ⁇ receptor
  • TBR2 type II TGF- ⁇ receptor
  • UMRC6 cells were derived from a clinically localized human renal cell carcinoma (Grossman et al., 1985). As shown in FIG. 14A , UMRC6 cells express type II TGF- ⁇ receptor (TBR2) mRNA, but not type III TGF- ⁇ receptor (TBR3). Immunohistochemical analysis ( FIG. 14B ) confirms the presence of TBR2 protein and the absence of TBR3 expression. UMRC3 cells were derived from the primary tumor of a patient with metastatic renal cell carcinoma. This highly aggressive cell line lacks detectable TBR2 and TBR3 mRNA ( FIG. 14A ) and protein ( FIG. 14B ).
  • TBR2 and TBR3 mRNA FIG. 14A
  • protein FIG. 14B
  • NRE normal renal epithelial
  • NRE cells can be grown in culture for 10 passages and were easily isolated and characterized. NRE cells were characterized for cytokeratin expression and tubule-specific gene expression, for example, megalin (data not shown). Thus, there are relevant cell models in which TBR2 and TBR3 expression can be manipulated to examine the impact of TGF- ⁇ receptor biology on the carcinogenesis and progression of human renal cell carcinoma in vitro.
  • TGF- ⁇ 1 inhibits cell proliferation in epithelial cells.
  • the present example demonstrates the effects of TGF- ⁇ on renal tumor cell proliferation.
  • DNA content of cells was used as a measure of cell proliferation.
  • Cells were plated at 20,000 cells/well in 12-well plates. Cells were grown in 10% FBS:DMEM:penicillin:streptomycin. The following day, media were exchanged with appropriate treatment added to the media. On day 3 of treatment, cells were analyzed for DNA content using Hoechst reagent. DNA standard was used to correlate DNA content per well. As shown in FIG. 15A (squares), TGF- ⁇ 1 inhibited the proliferation of normal renal epithelial cells in culture.
  • URMC3 cells expressed neither type II or type III TGF-t3 receptors and, not surprisingly, were resistant to the inhibitory effects of TGF- ⁇ on cell proliferation (triangles, FIG. 15A ).
  • UMRC6 cells expressed type II but not type III TGF- ⁇ receptors, and were partially resistant to TGF- ⁇ 1 (circles, FIG. 15A ).
  • TGF- ⁇ transcriptional activity was also measured in the above cell models using transient transfection of the 3TP/lux reporter, which contains an AP-1/Smad3 response element from the PAI-1 promoter.
  • This luciferase reporter construct demonstrates increased transcriptional activity in response to exogenous TGF- ⁇ -mediated signal transduction.
  • 3TP/lux was transiently transfected along with SV/renilla luciferase (Promega) into cells using fugene (Roche) as the transfection agent. Cells were treated with or without TGF- ⁇ 1 24 h after transfection and luciferase activity (Promega Luciferase Assay system and Lumat luminometer) was determined 24 h after TGF- ⁇ treatment.
  • Firefly luciferase activity was normalized using the ratio of firefly luciferase/renilla luciferase. As shown in FIG. 15B , normal renal epithelial cells were highly responsive to 2 ng/ml (80 pM) of TGF- ⁇ 1. UMRC6 cells demonstrated significantly less luciferase activity in response to TGF- ⁇ 1, and UMRC3 cells were entirely unresponsive.
  • UMRC3 cells were engineered to express stably either type II TGF- ⁇ receptor (+TBR2) alone or type II plus type III TGF- ⁇ eceptor (+TBR2+TBR3).
  • TBR2 human type II TGF- ⁇ receptor
  • the expression vector was stably transfected into UMRC3 cells using fugene as DNA carrier and genticin as selection antibiotic (Sigma, 1 mg/ml).
  • Ten clones (UMRC3/TBR2) were selected and verified for TBR 2 mRNA and protein expression such as Western analysis using the FLAG antibody (data not shown). From these cell clones, one was to be selected that had equivalent protein expression of TBR2 to that of normal renal epithelial (NRE) and UMRC6 cells.
  • TBR3 type III TGF- ⁇ receptor
  • pSV7d a gift from Dr C-H Heldin
  • TBR3 was then cloned into the EcoRI site of pcDNA4/TO/myc-His® (InVitrogen) in the sense and antisense (negative control) orientation. The orientation and sequence of TBR3 was verified.
  • the antisense TBR3 (As TBR3) vector was used as a control.
  • TBR3/pcDNA4/TO/myc-His and As TBR3/pcDNA4/TO/myc-His vectors were stably transfected into UMRC3/TBR2 cells.
  • a clone was selected that demonstrated an equivalent expression of TBR3 mRNA to that of normal renal epithelial cells.
  • wild-type UMRC3 were stably transfected with both pcDNA/FLAG and pcDNA4/TO/myc-His vectors.
  • TBR2 type II TGF- ⁇ receptor
  • TBR2+TBR3 type II plus type III TGF- ⁇ receptor
  • TGF-13-mediated transcriptional activity as a consequence of TGF- ⁇ receptor re-expression.
  • FIG. 16C reintroduction of TBR2 partially restored transcriptional responsiveness, as evidenced by a 5.6-fold increase in 3TP/lux activity after addition of TGF- ⁇ 1.
  • Reintroduction of both TBR2 and TBR3 into UMRC3 cells resulted in 17.5-fold increase in 3TP/lux activity after addition of TGF- ⁇ 1.
  • UMRC3 cells have been shown to be tumorigenic in athymic nude mice (Grossman et al., 1985). Anchorage independent growth in soft agar is a well-established in vitro correlate of in vivo tumorigenicity. Colonies formation in soft agar was determined as follows. UMRC3 (pcDNA/FLAG and pcDNA4/T0/myc-His empty vectors), UMRC3+TBR2, or UMRC3+TBR2+TBR3 cells were plated at 1000 cells/60 mm dish in an agarose/FBS/media sandwich in the presence of 2 ng/ml TGF- ⁇ . No selection antibodies were added to the agarose media mixture. The cells were incubated for 45 days to insure that no colony formation would occur. Cells were then stained with 0.005% Crystal Violet, photographed, and assessed for number and size of colonies.
  • UMRC3 cells demonstrated anchorage independent growth in soft agar.
  • Reintroduction of TBR2 into UMRC3 cells significantly decreased the number and size of colonies that formed in soft agar.
  • Reintroduction of both TBR2 and TBR3 completely abrogated the ability of UMRC3 cells to form colonies in soft agar, even after 45 days in culture.
  • TBR3 reintroduction of TBR3 in the presence of TBR2 into UMRC3 cells significantly enhanced TGF- ⁇ -regulated gene transcription, growth inhibition, and loss of anchorage-independent growth over that seen with reintroduction of TBR2 alone.
  • renal cell carcinoma cells are TGF- ⁇ resistant. Loss of TBR3 expression occurs early and appears to be associated with a relatively less aggressive state that is partially TGF- ⁇ responsive. Loss of TBR2 results in frank TGF- ⁇ resistance and is associated with acquisition of a more aggressive phenotype.
  • FIGS. 17-18 demonstrate that re-expression of type II or type III TGF- ⁇ receptor in the highly metastatic human renal cell carcinoma cell line UMRC3 inhibited cell proliferation in cell culture and tumor growth in a nude mouse model.
  • the TGF- ⁇ receptors were either re-expressed in a stable vector system or as an adenoviral vector.
  • TGF- ⁇ regulates a large number of diverse biological functions, including cell proliferation, differentiation, cell adhesion, apoptosis, extracellular matrix production, immune regulation, neuroprotection, and early embryonic development.
  • epithelial cells the effect of TGF- ⁇ is generally to inhibit proliferation, promote cellular differentiation, and regulate interactions with the extracellular matrix.
  • aberrations in TGF- ⁇ signaling can have a dramatic impact on cellular processes that are critically associated with neoplastic and malignant transformation.
  • TGF- ⁇ signaling is largely growth inhibitory, it makes intuitive sense that cancer cell would develop mechanisms to escape TGF- ⁇ sensitivity. To date, these mechanisms have not been elucidated in human renal cell carcinoma.
  • results presented in the present invention demonstrate that loss of type III TGF- ⁇ receptor expression is an early event in renal cell carcinoma biology and that this loss has important sequelae with regard to renal cell carcinoma carcinogenesis and progression.
  • All clinical samples of localized renal cell carcinoma demonstrated loss of type III TGF- ⁇ receptor, but had normal expression of type I and type II TGF- ⁇ receptors.
  • Replication of this clinical observation in in vitro models demonstrated significant loss of TGF- ⁇ sensitivity, manifest as a significant reduction in the growth inhibitory effects of TGF- ⁇ 1 and significantly reduced TGF- ⁇ -mediated transcription.
  • cell lines derived from localized RCC retained type II TGF- ⁇ receptor expression and therefore, still demonstrated sensitivity, albeit reduced, to TGF- ⁇ .
  • TGF- ⁇ receptor expression Only with metastatic progression and loss of type II TGF- ⁇ receptor expression does the cell become completely resistant to the effects of TGF- ⁇ . The investigators hypothesize that this retained, but attenuated, TGF- ⁇ signaling seen in local tumors must convey some as yet unrecognized biologic benefit for local tumors that is no longer required, and therefore discarded, with metastatic progression. In fact, this loss of type II TGF- ⁇ receptor expression may be an absolute integral component in the cascade of intracellular events that lead to the development of metastatic potential. In keeping with this hypothesis, it has been shown that loss of type I TGF-b receptor expression was one of 40 integral alterations of gene expression to predict for poor prognosis of patients diagnosed with renal cell carcinoma.
  • TBR3 reduced type III TGF- ⁇ receptor
  • TBR3 expression has been reported in human breast tumor cell lines, suggesting that loss of TBR3 expression may be a more ubiquitous phenomena in carcinogenesis, rather than an isolated finding in human RCC biology.
  • TBR3 plays an important functional role in signaling and that loss of expression is an important event in the acquisition of the tumorigenic and metastatic phenotype
  • FIG. 19 shows hierarchical clustering of genes over-expressed or down-regulated (with at least 2 fold differences) in stage I papillary renal cell carcinoma verses normal renal cortex. Genes over-expressed and down-regulated in stage I papillary renal cell carcinoma are listed in Table 8 and Table 9 respectively.
  • FIG. 20 shows hierarchical clustering of genes over-expressed or down-regulated (with at least 2 fold differences) in benign oncocytoma verses normal renal cortex. Genes over-expressed and down-regulated in benign oncocytoma are listed in Table 10 and Table 11 respectively.
  • FIG. 21 shows venn analysis of gene distribution among stage I renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma.
  • RRCC renal cell carcinoma
  • stage I RCC 95% confidence level
  • oncocytoma 95%
  • stage I papillary renal cell carcinoma 95% confidence level
  • FIG. 22 shows venn analysis of gene distribution among stage II renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma.
  • stage II RCC Genes with at least 2-fold differences in expression were filtered at 95% confidence level (CL) in the following 3 t-tests: stage II RCC vs. normal; oncocytoma vs. normal; and stage I papillary renal cell carcinoma vs. normal.
  • stage II RCC 95% confidence level
  • 152 genes were present only in oncocytoma (95% CL)
  • 334 genes were present only in stage I papillary renal cell carcinoma (95% CL)
  • 43 genes were common to stage II RCC, oncocytoma and stage I papillary renal cell carcinoma.

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Abstract

Gene expression profiling and hierarchical clustering analysis readily identify differential gene expressions in normal renal epithelial cells and renal cell carcinomas. Genes identified by this analysis would be useful for diagnosis, prognosis and development of targeted therapy for the prevention and treatment of conventional renal cell carcinoma.

Description

    CROSS-REFERENCE TO RELATED APPLICATION
  • This is a continuation application under 35 U.S.C. §120 of pending nonprovisional application U.S. Ser. No. 10/938,973, filed Sep. 10, 2004, which claims benefit of provisional application U.S. Ser. No. 60/539,838, filed Jan. 28, 2004, now abandoned, and of provisional application U.S. Ser. No. 60/502,038, filed Sep. 10, 2003, now abandoned, the entirety of all of which are hereby incorporated by reference.
    • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • The present invention relates generally to the field of cancer research. More specifically, the present invention relates to gene expression profiling for human renal cell carcinoma.
  • 2. Description of the Related Art
  • Renal cell carcinoma (RCC) represents a major health issue. The American Cancer Society predicts 31,900 new cases will be diagnosed in the United States alone in the year 2003, with 11,900 people dying of the disease. When clinically localized or even locally advanced, renal cell carcinoma can be surgically resected for cure using a variety of approaches. With metastatic progression, however, renal cell carcinoma is incurable, and existing systemic therapies are largely ineffective in impacting disease response or patient survival. The lack of effective systemic therapy for metastatic renal cell carcinoma is, in part, due to a fundamental lack of understanding of the molecular events that result in cellular transformation, carcinogenesis, and progression in human kidney.
  • The advent of gene array technology has allowed classification of disease states at molecular level by examining changes in all mRNAs expressed in cells or tissues. Gene expression fingerprints representing large numbers of genes may allow precise and accurate grouping of renal cell carcinoma. Moreover, large scale gene expression analysis have the potential of identifying a number of differentially expressed genes in renal cell carcinoma compare to normal renal epithelial cells. These genes or markers may further be tested for clinical utility in the diagnosis and treatment of renal cell carcinoma.
  • Thus, the identification of novel renal cell carcinoma markers to be used for detection, diagnosis and development of effective therapy against the disease remains a high priority. The prior art is deficient in understanding the molecular differences between renal cell carcinoma and normal renal epithelium. The present invention fulfills this need in the art by providing gene expression profiling for these two types of tissues.
    • SUMMARY OF THE INVENTION
  • The present invention identifies genes with a differential pattern of expression between different subtypes of renal cell carcinomas (RCC) and normal renal epithelium. These genes and their products can be used to develop novel diagnostic and therapeutic markers for the treatment of renal cell carcinomas.
  • Genomic profiling of conventional renal cell carcinoma tissues and patient-matched normal kidney tissue samples was carried out using stringent statistical analyses (ANOVA with full Bonferroni corrections). Subtypes of renal cell carcinoma include stage I, II, III, and IV (reflecting differences in tumor size, lymph node and organ metastasis), stage I papillary renal cell carcinoma, and benign oncocytoma. Hierarchical clustering of the expression data readily distinguished normal tissue from renal cell carcinomas. The identified genes were verified by real-time FCR and immunohistochemical analyses.
  • Different subtypes of conventional renal cell carcinomas can be diagnosed either by drawing blood and identifying secreted gene products specific to renal cell carcinoma or by doing a biopsy of the tissue and then identify specific genes that are altered when renal cell carcinoma is present. An example of when this may be especially important is in distinguishing the deadly conventional renal cell carcinomas (account for 85% of all renal cell carcinomas) from renal oncocytoma (benign renal cell carcinoma) as well as identifying the histologic subtypes of papillary and sarcomatoid renal cell carcinoma. Identification of specific genes will also help in determining which patients will have a good prognosis verses that of a poor prognosis. In addition, subsets of genes identified in the present invention can be developed as targets for therapies that could cure, prevent, or stabilize the disease. Thus, results from the present invention could be used for diagnosis, prognosis, and development of therapies to treat or prevent renal cell carcinoma.
  • In one embodiment, there are provided methods of detecting conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein. In another embodiment, conventional or clear cell renal cell carcinoma is detected based on decreased expression of type III TGF-β receptor.
  • In yet another embodiment, there are provided methods of detecting stage I conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
  • The present invention also provides methods of detecting stage II conventional or clear cell renal cell carcinoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
  • The present invention also provides methods of detecting papillary renal cell carcinoma or benign oncocytoma based on over-expression and/or down-regulation of a number of genes disclosed herein.
  • In another embodiment, there is provided a method of targeting conventional or clear cell renal cell carcinoma cells based on generating antibodies or small molecules directed against a cell surface molecule over-expressed in conventional renal cell carcinoma cells.
  • In yet another embodiment, there is provided a method of treating conventional or clear cell renal cell carcinoma by replacing down-regulated tumor suppressor gene in conventional renal cell carcinoma.
  • Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1A shows hierarchical clustering of genes expressed in normal renal cortex (12 patient tissue samples) verse stage I conventional renal cell carcinoma (6 patient tissue samples). Red indicates that a gene is highly expressed and green is indicative of low expression. Four hundred eighty eight genes were depicted in FIG. 1A. FIG. 1B shows hierarchical clustering of genes expressed in normal renal cortex (12 patient tissue samples) verse stage II conventional renal cell carcinoma (6 patient tissue samples). Red indicates that a gene is highly expressed and green is indicative of low expression. Six hundred twenty eight genes were depicted in FIG. 1B. FIG. 1C shows hierarchical clustering of genes selected from a Venn analysis in which the chosen genes were expressed in common in both stage I and II at a 99% confidence level. One hundred eighty eight genes were depicted in FIG. 1C. C, cancer cells; N, normal cells; S1, stage 1; S2, stage 2.
  • FIG. 2 shows TGF-β1 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR TGF-β1 mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
  • FIG. 3 shows TGF-α mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF-α mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
  • FIG. 4 shows adrenomedulin mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. Adrenomedulin mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
  • FIG. 5 shows TGF-tβ2 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF-β32 mRNA levels were not altered between normal and tumor matched samples.
  • FIG. 6 shows TGF-133 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. TGF-β3 mRNA levels were not altered between normal and tumor matched samples.
  • FIG. 7 shows tumor suppressor gene Wilms Tumor 1 (WT1) mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. WT1 mRNA levels were down-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts.
  • FIG. 8 shows von Hippel Lindau mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. A small percentage of tumor tissues demonstrated attenuated von Hippel Lindau mRNA levels when compared to matched normal tissue
  • FIG. 9 shows calbindin mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. Calbindin mRNA was completely lost in all stage I renal cell carcinoma. p<0.05 compared to matched control. *Stage I tumor: 0±0; stage III tumor: 0.0009±0.0004; stage IV tumor: 0.003±0.0004/
  • FIG. 10 shows MUC1 mRNA expression in stages I-IV renal cell carcinoma as measured by real time PCR. MUC1 mRNA levels were down-regulated in all tumor tissues as early as stage I. *p<0.05 compared to matched control.
  • FIGS. 11A-11B show stepwise loss of type III α receptor (TBR3) and type II TGF-β receptor (TBR2) mRNA expression during renal cell carcinogenesis and progression in patient tissue samples. FIG. 11A shows gene array data from 10 patients—five diagnosed with localized renal cell carcinoma and five with metastatic disease. ‘+’ (P<0.05) indicates statistical difference for TBR3 mRNA levels as compared to normal tissue and ‘*’ (P<0.28) indicates statistical difference for TBR2 mRNA levels as compared to normal controls. Data are expressed as mean±s.e. FIG. 11B shows real-time RT-PCR verification of TBR1, TBR2, and TBR3 mRNA levels of tissue samples described in FIG. 11A. Data are expressed as mean±s.d.
  • FIG. 12 shows immunohistochemistry of patient tissue demonstrating loss of type III α receptor (TBR3) expression (top row) in all tumors, loss of type II α receptor (TBR2) expression (middle row) in patients diagnosed with metastatic tumors, and no change in type I α receptor (TBR1) protein expression (bottom row).
  • FIG. 13 demonstrates down-regulation of TGF-3-regulated genes in human tumor tissues by real-time PCR. Genes known to be up-regulated by α are suppressed in tumor tissues. Down-regulation of collagen IV type 6, fibulin 5, and connective tissue growth factor (CTGF) mRNA in tumor tissues were compared to matched normal tissue controls. Values were normalized to 18 s mRNA. Each matching tumor value was compared to its respective normal control. The mean±s.d. was calculated for each sample group with n values of 10-15 matched samples.
  • FIGS. 14A-14B show tumor cell lines that lose type III α receptor (TBR3) and type I TGF-β receptor (TBR2) expression. FIG. 14A shows semi-quantitative RT-PCR measurements of mRNA levels of TBR1, TBR2, and TBR3 for UMRC3, UMRC6 and normal renal epithelial (NRE) cells. FIG. 14B shows immunohistochemistry of protein expression for TBR1, TBR2, and TBR3 (×40 magnification).
  • FIGS. 15A-15B show loss of type III TGF-β receptor (TBR3) and type II α receptor (TBR2) expression in renal tumor cell lines correlate with loss of TGF-3-regulated growth inhibitory and transcriptional responses. FIG. 15A shows cell proliferation was inhibited as assessed by DNA content 3 days after α treatment. Percent of each respective untreated control was used for comparisons. Transient transfection using 3TP/lx along with a renilla luciferase control demonstrates loss of responsiveness to 2 ng/ml TGF-β1 with loss of TGF-β receptor expression (FIG. 15B). Firefly luciferase activity was normalized using the ratio of firefly luciferase/renilla luciferase. Data are expressed as mean±s.d.
  • FIG. 16A demonstrates RT-PCR derived mRNA expression of type III α receptor (TBR3), type II α receptor (TBR2), and type I α receptor (TBR1) in UMRC3 cells and cells stably transfected with TBR2 and TBR3. FIG. 16B shows UMRC3 cells stably transfected with type II TGF-β receptor (UMRC3+TBR2) or type II and type III TGF-β receptor (UMRC3+TBR2+TBR3) demonstrated attenuated cell proliferation following the administration of exogenous TGF-β1 as compared to that of UMRC3 cells. FIG. 16C shows UMRC3 cells, UMRC3+TBR2 cells, and UMRC3+TBR2+TBR3 stable cell lines transfected with 3TP/lux were treated with or without TGF-β and examined for luciferase activity. FIG. 16D shows real-time PCR measuring mRNA levels for collagen IV type 6 in UMRC3, UMRC3+TBR2 cells, and UMRC3+TBR2+TBR3 cells in the presence of 2 ng/ml TGF-β1 for 24 h. FIG. 16E shows colony formation assay demonstrates that UMRC3+TBR2+TBR3 cells have completely lost anchorage-independent growth, while attenuated growth in UMRC3+TBR2 cells occurs as compared to that of UMRC3 cells. The number of colonies were stained and counted after 45 days of growth. Data are expressed as mean±s.d.
  • FIG. 17A shows growth inhibition after re-expressing human type III TGF-β3 receptor (TBR3) in UMRC3 cells. UMRC3 cells were stably transfected with TBR3 or infected using an adenoviral vector expressing TBR3. Cells were plated in culture dishes at 20,000 cells/well. Cell number was determined at the indicated times using a Coulter cell counter. FIG. 17B shows RT-PCR data demonstrating the mRNA expression levels of type I, II, or III TGF-β receptors (TBR1, TBR2, TBR3) in UMRC3 cells in the presence or absence of the adenoviral vector expressing TBR3. Unmodified UMRC3 cells only express TBR1.
  • FIG. 18 shows re-expression of human type II or III TGF-β receptors (TBR2 or TBR3) inhibits tumor growth in nude mice. One million UMRC3 cells stably transfected with human type II or type III TGF-β receptors were implanted into nude mice ectopically and tumor growth was measured weekly. Tumor volume (mm3) was calculated by width×length×height×0.5236.
  • FIG. 19 shows hierarchical clustering of genes expressed in normal renal cortex verse stage I papillary renal cell carcinoma. Red indicates that a gene is highly expressed and green is indicative of low expression.
  • FIG. 20 shows hierarchical clustering of genes expressed in normal renal cortex verse benign oncocytoma. Red indicates that a gene is highly expressed and green is indicative of low expression.
  • FIG. 21 shows venn analysis of gene distribution among stage I renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma.
  • FIG. 22 shows venn analysis of gene distribution among stage II renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • High-throughput technologies for assaying gene expression, such as high-density oligonucleotide and cDNA microarrays, offer the potential to identify clinically relevant genes differentially expressed between normal and tumor cells. The present invention discloses a genome-wide examination of differential gene expression between renal cell carcinomas (RCC) and normal renal epithelial cells.
  • Currently, there are no proven molecular markers useful clinically for the diagnosis, staging, or prognosis of sporadic renal cell carcinoma. The present invention detects genes that have differential expression between renal cell carcinomas and normal renal epithelial cells. The known function of some of these genes may provide insight into the biology of renal cell carcinomas while others may prove to be useful as diagnostic or therapeutic markers against renal cell carcinomas. Subtypes of renal cell carcinomas disclosed herein include stage I, II, III, and IV renal cell carcinomas (reflecting differences in tumor size, lymph node and organ metastasis), stage I papillary renal cell carcinoma, and benign oncocytoma.
  • The present invention provides methods of detecting conventional renal cell carcinoma based on determining the expression level of a number of genes that are found to have 2-fold or higher differential expression levels between tumor and normal tissue. In general, biological samples (e.g. tissue samples, serum samples, urine samples, saliva samples, blood samples or biopsy samples) are obtained from the individual to be tested and gene expression at RNA or protein level is compared to that in normal tissue. The normal tissue samples can be obtained from the same individual who is to be tested for renal cell carcinoma.
  • It will be obvious to one of ordinary skill in the art that gene expression can be determined by DNA microarray and hierarchical cluster analysis, real-time PCR, RT-PCR, or northern analysis, whereas secreted gene products can be measured in blood samples by standard procedures.
  • In one embodiment, there is provided a method of detecting conventional or clear cell renal cell carcinoma based on differential expression of one or more of the following genes or proteins: TGF-β1, TGF-α, adrenomedulin, fibroblast growth factor 2 (FGF2), vascular epidermal growth factor (VEGF), osteonectin, follistatin like-3, inhibin beta A, spondin 2, chemokine X cytokine receptor 4 (CXCR4), fibronectin, neuropilin 1, frizzled homolog 1, insulin-like growth factor binding protein 3, laminin alpha 3, integrin beta 2, semaphorins 6A, semaphorins 5B, semaphorins 3B, caspase 1, sprouty 1, CDH16, PCDH9, compliment component 1-beta, compliment component 1-alpha, compliment component 1-gamma, CD53, CDW52, CD163, CD14, CD3Z, CD24, RAP1, angiopoietin 2, cytokine knot secreted protein, MAPKKKK4, 4-hydroxyphenylpyruvate dioxygenase, pyruvate carboxyknase 2, 11-beta-hydroxysteroid dehydrogenase 2, GAS1, CDKN1, nucleolar protein 3, interferon induced protein 44, NR3C1, vitamin D receptor, hypothetical protein FLJ14957 (Affy #225817_at), metallothionein 2A, metallothionein-If gene, metallothionein 1H, secreted frizzled related protein 1, connective tissue growth factor, and epidermal growth factor.
  • In another embodiment, there is provided a method of detecting conventional renal cell carcinoma by examining the expression level of type III TGF-β receptor, wherein decreased expression of type III TGF-b receptor indicates the presence of renal cell carcinoma. In general, the expression level of type III TGF-β receptor can be determined at the mRNA or protein level.
  • The present invention also provides methods of detecting stage I conventional renal cell carcinoma, stage II conventional renal cell carcinoma, stage I papillary renal cell carcinoma, or benign oncocytoma based on over-expression or down-regulation of a number of genes identified in the present invention. The present invention discloses a number of genes that are up- or down-regulated specifically in these subtypes of renal cell carcinoma. Determining the expression levels of these genes would provide specific diagnosis for these different subtypes of renal cell carcinoma.
  • For example, stage I conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 1, (ii) down-regulation of one or more genes listed in Table 2, or (iii) over-expression of one or more genes listed in Table 1 and down-regulation of one or more genes listed in Table 2. Similarly, stage II conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 3, (ii) down-regulation of one or more genes listed in Table 4, or (iii) over-expression of one or more genes listed in Table 3 and down-regulation of one or more genes listed in Table 4.
  • The present invention also discloses a number of genes that are up- or down-regulated in both stage I and stage II conventional renal cell carcinoma (Tables 5 and 6 respectively). These genes would also provide diagnosis for stage I or stage II conventional renal cell carcinoma. Hence, stage I or stage II conventional renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 5, or (ii) down-regulation of one or more genes listed in Table 6.
  • In another embodiment, stage I papillary renal cell carcinoma can be detected based on (i) over-expression of one or more genes listed in Table 8, (ii) down-regulation of one or more genes listed in Table 9, or (iii) over-expression of one or more genes listed in Table 8 and down-regulation of one or more genes listed in Table 9.
  • In yet another embodiment, benign oncocytoma can be detected based on (i) over-expression of one or more genes listed in Table 10, (ii) down-regulation of one or more genes listed in Table 11, or (iii) over-expression of one or more genes listed in Table 10 and down-regulation of one or more genes listed in Table 11.
  • In still yet another embodiment, there are provided methods of utilizing genes over-expressed on the cell surface of renal carcinoma tissue to develop antibodies or other small molecules for the purpose of specifically targeting the renal tumor cells. The present invention discloses a number of genes that are up-regulated in stage I renal cell carcinoma (RCC), stage II RCC tumor, stage I papillary RCC, and benign oncocytoma. Antibodies or small molecules directed against proteins encoded by these genes can be linked with a therapeutic drug to deliver drug to the tumor tissue, or be linked with dye, nanoparticle or other imaging agents for cancer imaging. Some of the novel genes identified herein for the first time include, but are not limited to, the following genes: calcitonin receptor-like (206331_at; 210815_s_at); receptor (calcitonin) activity modifying protein 2 (RAMP2; 205779_at); endothelin receptor type B (206701_x_at); beta 2 integrin (202803_s_at); alpha 5 integrin (201389_at); chemokine X cytokine receptor 4 (CXCR4); fibronectin; neuropilin 1 (212298_at; 210510_s_at); CD24; CD14; Cd163; CD53; Compliment Componenet 1-beta, 1-alpha, and 1-gamma; CDH4; integrin beta2; ADAM28; FK506 binding protein; collagen Valpha2; tumor necrosis factor receptor superfamily, member 6; tumor necrosis factor receptor superfamily, member 5; tumor necrosis factor (ligand) superfamily, member 13b; tumor necrosis factor receptor superfamily, member 12A; and the FGF receptor.
  • In another embodiment, there is provided a method of treating conventional or clear cell renal cell carcinoma. The method involves replacing tumor suppressor genes (e.g., via gene therapy) whose expression is down-regulated in tumor tissues or introducing a molecule that induces the down-regulated gene to be re-expressed in the tumor. The present invention discloses a number of genes that are down-regulated in stage I renal cell carcinoma (RCC), stage II RCC tumor, stage I papillary RCC, and benign oncocytoma. Some examples of down-regulated genes identified in stage I and/or II RCC tumors include, but are not limited to, CDKN1, secreted frizzled related protein 1, semaphoring 6D, semaphoring 3B, CDH16, TNF alpha, calbindin D28, defensin beta1, beta-catenin interacting protein 1, GAS1, vitamin D receptor, Kruppel-like factor 15. This method of treatment can be combined with other therapies to provide combinatorial therapy.
  • The genes that are found to have altered expression in stage I and stage II renal cell carcinoma would also be useful for determining patient prognosis. These genes or gene products (i.e., proteins) would have the unique characteristic of being altered in tumor verses normal samples in a subset of patients. For example, basic transcription element binding protein 1 is down-regulated in 7 out of 12 renal cell carcinoma tumors. Other examples include CD164, decreased 5/12; Map kinase kinase kinase 7, increased 6/12; Endoglin, increased 7/12; SERPIN A1, increased 6/12; Metalloprotease 11 (MMP11), increased 7/12; Integrin 3 alpha, increased 4/12; carbonic anhydrase II, decreased 7/12; protein tyrosine kinase 2, increased 4/12; fibroblast growth factor 11, increased 6/12; fibroblast growth factor 2, increased 7/12; VEGF B, increased 5/12.
  • Moreover, the levels of change may be a useful determinant of patient outcome and/or rationale for strategy of treatment course. An example of this is found for chemokine (C-X-C motif) ligand 14 (CXCL14, 222484_s_at). Six patients with stage I and six patients with stage II renal cell carcinoma were analyzed by genomic profiling. A patient with stage I renal cell carcinoma has CXCL14 mRNA expression levels of 19862 and 24.49 in his normal tissue and tumor tissue respectively. This patient would be predicted to have a poor prognosis or poor response to therapy based upon this result along with other gene predictors. On the other hand, a patient with stage II RCC has CXCL14 mRNA expression levels of 20435 and 18557 in his normal tissue and tumor tissue respectively. This patient would be predicted to have a good prognosis and good response to chemotherapy.
  • The following examples are given for illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
    • Example 1 Tissue Banking
  • Renal tissue (normal and tumor) was transported to a sterile hood on ice and under sterile conditions. Tissue was dissected under the direction of a pathologist. The tissue was frozen in liquid nitrogen for isolation of RNA, DNA, and protein or processed to establish primary cell cultures. The tissue was fixed in formalin for immunohistochemistry and in situ hybridization and RNAlater (Ambion) for RNA isolation. Primary normal renal epithelial (NRE) cell cultures were established using standard collagenase/Dnase techniques to digest tissue and isolate single cells. NREs were easily isolated and grew well in culture for up to 10 passages. These cells were further analyzed for homogeneity with regard to epithelial population using appropriate immunohistochemical markers such as vimentin, cytokeratin, and megalin.
    • Example 2 Genomic Gene Array and Microarray Data Analysis
  • Gene expression profiling was performed using Affymetrix HU95A oligonucleotide gene arrays (>12,600 genes) or HG-U133 A&B GeneChip® oligonucleotide microarrays (33,000+ probe sets). Total RNA (Trizol®, Ambion) was extracted from patient-matched normal renal cortex and tumor tissue from patients diagnosed with local disease confined to the kidney. Alternatively, the investigators analyzed metastatic disease defined by lesions in lymph nodes, adrenal, or other organs. Data were analyzed by a combination of two-dimensional ANOVA, Affymetrix MAS5.0®, and hierarchical cluster analysis using Spotfire®. Procedure that were used to identify altered expression of large sets of genes, as well as other issues concerning microarray analyses can be found in a recent review article by Copland et al. (2003).
    • Example 3 Real-Time PCR
  • Applied Biosystems' assays-by-design or assays-on-demand 20× assay mix of primers and TaqMan® MGB probes (FAM® dye-labeled) for all target genes and predeveloped 18S rRNA WIC® dye-labeled probe) TaqMan® assay reagent for internal control were used for real-time PCR measurements. These assays were designed to span exon-exon junctions so as not to detect genomic DNA and all primers and probes sequences were searched against the Celera database to confirm specificity. Validation experiments were performed to test the efficiency of the target amplification and the efficiency of the reference amplification. All absolute values of the slope of log input amount versus DCT is less than 0.1.
  • Separate tubes (singleplex) for one-step RT-PCR was performed with 50 ng RNA for both target genes and endogenous controls using TaqMan® one-step RT-PCR master mix reagent kit (Applied Biosystems). The cycling parameters for one-step RT-PCR were: reverse transcription 48° C. for 30 min, AmpliTaq® activation 95° C. for 10 min, denaturation 95° C. for 15 s, and annealing/extension 60° C. for 1 min (repeat 40 times) on ABI7000®. Duplicate CT values were analyzed with Microsoft Excel® using the comparative CT(DDCT) method as described by the manufacturer (Applied Biosystems). The amount of target (2−DDCT) was obtained by normalizing to an endogenous reference (18smRNA) and relative to a calibrator (normal tissue).
    • Example 4 Immunohistochemical Analyses of Protein Expression
  • For immunohistochemical analyses of type I TGF-β receptor (TBR1), type II TGF-β receptor (TBR2), and type III TGF-β receptor (TBR3) expression, patient-matched normal renal and tumor tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks. Consecutive sections were cut 5 um thick, deparaffinized, hydrated, and immunostained using antibodies recognizing human TBR1, TBR2, and TBR3 (1:100; Santa Cruz Biotechnology). Biotinylated secondary antibody (1:600; Santa Cruz Biotechnology) was detected using avidin-biotin-peroxidase detection according to the manufacturer's instructions (Vectastatin Elite ABC kit; Vector Lab). All slides were lightly counterstained with hematoxylin before dehydration and mounting.
  • For cell lines, cells were plated on glass coverslips in wells. Prior to the detection of TGF-β receptor expression as described above, cells were fixed onto the coverslips with 3% formalin.
    • Example 5 Gene Expression Profiling of Renal Cell Carcinoma
  • Gene expression profiling was performed using Affymetrix oligonucleotide gene arrays. RNA was extracted from patient-matched normal renal cortical and tumor tissues from patients diagnosed with localized and metastatic renal cell carcinoma. Data were analyzed by a combination of two-dimensional ANOVA, Affymetrix MAS5.0®, and hierarchical cluster analysis using Spotfire® (reviewed in Copland et al., 2003).
  • A primary goal of microarray analysis is to discover hidden patterns of differential expression within a large data field. Normal renal cortical and primary tumor tissue with no metastasis were collected from patients diagnosed with local disease. Normal tissue, primary tumor, and metastatic tissue were also collected from patients diagnosed with metastatic disease. Comparison of patient-matched normal and tumor tissue allowed for the discovery of changes in mRNA levels between normal and tumor tissue, as well as local and metastatic disease.
  • Heatmaps with two-way dendograms depicting genes specifically altered in tumor tissue as compared to normal renal cortex are shown in FIG. 1. FIG. 1A shows hierarchical clustering of genes expressed in normal renal cortex verses stage I conventional renal cell carcinoma. FIG. 1B shows hierarchical clustering of genes expressed in normal renal cortex verses stage II renal cell carcinoma. FIG. 1C shows hierarchical clustering of genes selected from a Venn analysis in which the chosen genes were expressed in common in both stage I and II at a 99% confidence level.
  • TGF-β1, TGF-α and adrenomedulin mRNA levels were up-regulated in all stages of renal cell carcinoma as compared to normal tissue counterparts (FIGS. 2-4), whereas TGF-β2 and TGF-β3 mRNA levels were not altered between normal and tumor matched samples (FIGS. 5-6).
  • Tumor suppressor gene Wilms Tumor 1 (WT1) was down-regulated in all stages of renal cell carcinoma (FIG. 7). A small percentage of tumor tissues demonstrated attenuated von Hippel Lindau mRNA levels when compared to matched normal tissue (FIG. 8). Calbindin mRNA was completely lost (FIG. 9) while MUC1 was greatly attenuated in stage I renal cell carcinoma (FIG. 10).
  • The present analysis identifies 278 genes that were up-regulated in stage I renal cell carcinoma, whereas 380 genes were up-regulated in stage II renal cell carcinoma. Among these genes, 82 were up-regulated in both stages I and II renal cell carcinoma. One hundred fifty nine genes were down-regulated in stage I renal cell carcinoma, whereas 195 genes were down-regulated in stage II RCC. Among these genes, 82 were down-regulated in both stage I and II renal cell carcinoma.
  • Genes over-expressed and down-regulated in stage I renal cell carcinoma are listed in Table 1 and Table 2 respectively. Genes over-expressed and down-regulated in stage I renal cell carcinoma are listed in Table 3 and Table 4 respectively. Genes over-expressed in both stage I and II renal cell carcinoma are listed in Table 5. Genes down-regulated in both stage I and II renal cell carcinoma are listed in Table 6.
  • TABLE 1
    Genes With Up-Regulated Expression In stage I Renal Cell Carcinoma
    Genbank ID Gene Symbol Genbank ID Gene Symbol
    NM004356.1 CD81 NM004079.1 CTSS
    NM002293.2 LAMC1 NM001784.1 CD97
    NM000980.1 RPL18A AF151853.1 PREI3
    AK002091.1 MGEA5 NM000491.2 C1QB
    NM005721.2 ACTR3 BC000125.1 TGFB1
    NM002668.1 PLP2 NM004520.1 KIF2
    NM021038.1 MBNL NM000321.1 RB1
    AF070656.1 YME1L1 NM012262.2 HS2ST1
    NM021029.1 RPL36A NM000560.1 CD53
    NM002945.1 RPA1 NM005502.1 ABCA1
    NM002480.1 PPP1R12A AF285167.1 ABCA1
    NM001349.1 DARS BG170541 MET
    NM005496.1 SMC4L1 NM021642.1 FCGR2A
    AW163148 MARCKS BE967532 KIAA0220
    NM002356.4 MARCKS NM006526.1 ZNF217
    M68956.1 MARCKS NM000570.1 FCGR3B
    AI589086 LAPTM5 N26005 PPP1R3C
    NM006762.1 LAPTM5 NM006153.1 NCK1
    NM014267.1 SMAP NM001549.1 IFIT4
    NM000235.1 LIPA NM003141.1 SSA1
    NM000176.1 NR3C1 NM014705.1 KIAA0716
    NM005737.2 ARL7 NM005197.1 CHES1
    NM005737.2 ARL7 NM002907.1 RECQL
    BC001051.1 ARL7 U43328.1 CRTL1
    NM006169.1 NNMT NM017925.1 FLJ20686
    NM005862.1 STAG1 NM006773.2 DDX18
    AI356412 LYN U20350.1 CX3CR1
    NM002350.1 LYN NM005761.1 PLXNC1
    BG107456 TRIP-Br2 NM004834.1 MAP4K4
    NM021913.1 AXL NM021644.1 HNRPH3
    NM002194.2 INPP1 NM006640.1 MSF
    NM019058.1 RTP801 NM004180.1 TANK
    NM002110.1 HCK AW148801 NAP1L1
    NM030755.1 TXNDC AB011118.1 KIAA0546
    NM030984.1 TBXAS1 AU145005 SP3
    NM014350.1 GG2-1 N80918 CG018
    BC001312.1 P5 BF439472 ATP11A
    U14990.1 RPS3 BE968801 RPL35A
    D83043.1 HLA-B AI985751 NAP1L1
    AI888672 NAP1L1 AI735692 LST1
    BC002387.1 NAP1L1 AA995910 ALOX5
    M60334.1 HLA-DRA M12679.1 HUMMHCW1A
    AF161522.1 C3orf4 AL133053.1 FLJ23861
    BG256677 IFI16 X03348.1 NR3C1
    M26880.1 UBC AC005339 N/A
    U17496.1 PSMB8 AK024836.1 HLA-C
    AF141347.1 TUBAS AC003999 SCAP2
    L01639.1 CXCR4 AJ224869 CXCR4
    NM005445.1 CSPG6 AL022067 PRDM1
    AB030655.1 EFEMP2 AL110158.1 KIAA1078
    AF165520.1 APOBEC3C S81916.1 N/A
    AF009670.1 ABCC3 M80469 N/A
    AF020314.1 CMRF-35H NM002860.1 PYCS
    BC001606.1 NCF2 NM020198.1 GK001
    BC005352.1 GG2-1 NM016304.1 C15orf15
    AF281030.1 HRIHFB2122 AA102574 BAZ1A
    BC001052.1 RECQL NM024844.1 PCNT1
    L32610.1 HNRPH3 NM015938.1 CGI-07
    M23612.1 RASA1 NM018200.1 HMG20A
    AF109683.1 LAIR1 NM025235.1 TNKS2
    BC002841.1 HSA9761 NM015991.1 C1QA
    D29640.1 IQGAPI NM016090.1 RBM7
    L25259.1 CD86 NM024554.1 PGBD5
    M60333.1 HLA-DRA NM017718.1 FLJ20220
    U13698.1 CASP1 NM017923.1 FLJ20668
    U90940.1 FCGR2C NM030921.1 DC42
    M90685.1 HLA-G BC004470.1 ASC
    M90684.1 HLA-G AK021413.1 LARS
    M90686.1 HLA-G BF444916 FAD104
    L22453.1 RPL3 BC004819.1 PLDN
    U01351.1 NR3C1 AF247167.1 AD031
    U62824.1 HLA-C U39402.1 N/A
    L07950.1 HLA-B BC006112.1 DKFZP434B195
    AF348491.1 CXCR4 BG388615 N/A
    NM003079.1 SMARCEI AB033007.1 KIAA1181
    BE646386 EXO70 BG250721 N/A
    A1972475 N/A AK024221.1 C40
    AA195999 MAPK1 BF477658 N/A
    AL049397.1 N/A BG251556 KIAA1949
    BE895685 KIAA0853 AB033091.1 KIAA1265
    M82882.1 ELF1 AK024350.1 AMOTLI
    AB020633.1 KIAA0826 NM018440.1 PAG
    AL031781 N/A AW500180 N/A
    BF209337 MGC4677 AW026543 N/A
    AI709406 N/A AI092770 N/A
    AI806905 N/A NM020679.1 AD023
    AI392933 FLJ36090 AK024855.1 CTSS
    AI142096 N/A AK000119.1 N/A
    AL137430.1 N/A AW977527 PRDM1
    AV724266 FLJ20093 BE671060 N/A
    BF589359 N/A AL037450 N/A
    AW084125 CAPZA1 AI401535 N/A
    N20927 RAP2B AV683852 N/A
    AI627666 LOC115548 BF055144 N/A
    AV726322 N/A AA352113 N/A
    AI697657 LANPL BF056209 N/A
    BF002625 N/A X60592 TNFRSF5
    BF439533 N/A
  • TABLE 2
    Genes With Down-Regulated Expression
    In stage I Renal Cell Carcinoma
    Genbank ID Gene Symbol
    L38487 ESRRA
    NM004415.1 DSP
    NM005327.1 HADHSC
    NM003321.1 TUFM
    NM002084.2 GPX3
    AI983043 N/A
    NM006066.1 AKR1A1
    NM006384.2 CIB1
    NM001685.1 ATP5J
    NM014652.1 IMP13
    NM013410.1 AK3
    NM016725.1 FOLR1
    NM021151.1 CROT
    NM005951.1 MT1H
    NM005952.1 MT1X
    AL080102.1 N/A
    BC000931.2 ATP5C1
    BC005398.1 DKFZP566D193
    D87292.1 TST
    AU151428 IDH2
    BC000109.1 ILVBL
    AF333388.1 N/A
    NM005953.1 MT2A
    BF217861 N/A
    AA594937 COBL
    AW052179 COL4A5
    AI884867 LOC155066
    BF246115 N/A
    AW028110 KIAA0500
    AW242315 N/A
    AW080549 FUT3
    AW149846 GPX3
    AI038402 N/A
    AI051046 MGC4614
    AI659456 N/A
    AW664964 N/A
    AI631895 SGK2
    AI263078 FLJ31168
    BF057634 HOXD8
    AA746038 GPR110
    AK024386.1 GRHPR
    AL109716.2 N/A
    AK026411.1 ALDOB
    M10943 N/A
    AW088547 N/A
    NM018049.1 GNRPX
    NM017900.1 AKIP
    NM006548.1 IMP-2
    NM025135.1 KIAA1695
    NM016458.2 LOC51236
    NM022128.1 RBSK
    NM015974.1 CRYL1
    NM013333.1 EPN1
    AA133341 C14orf87
    AF226732.1 NPD007
    AF265439.1 MRPS15
    AI743534 DKFZP564B1162
    AB042647.1 B29
    AL522667 ORF1-FL49
    BG255416 KIAA0114
    AF308301.1 MRPS26
    BE408081 N/A
    AL521634 FLJ32452
    BF203664 MGC14288
    BE645551 MGC39329
    AW193698 TGFBR3
    BF540829 N/A
    W72455 FLJ25476
    AI457453 N/A
    BF056892 N/A
    AK024386.1 GRHPR
    AL109716.2 N/A
    AA442776 N/A
    AI913600 N/A
    AW771908 N/A
    AI807887 N/A
    AW102941 N/A
    AW024656 N/A
    AB002342 PRKWNK1
  • TABLE 3
    Genes With Up-Regulated Expression
    In stage II Renal Cell Carcinoma
    Genbank ID Gene Symbol
    NM006096.1 NDRG1
    NM006098.1 GNB2L1
    NM001780.1 CD63
    NM003118.1 SPARC
    NM000291.1 PGK1
    NM003870.1 IQGAP1
    AB032261.1 SCD
    NM002629.1 PGAM1
    NM003564.1 TAGLN2
    NM000310.1 PPT1
    NM003405.1 YWHAH
    U82164.1 MIC2
    NM002305.2 LGALS1
    NM001096.1 ACLY
    NM002121.1 HLA-DPB1
    NM021038.1 MBNL
    NM003651.1 CSDA
    AV685920 CAPZA2
    NM002654.1 PKM2
    NM001175.1 ARHGDIB
    BC000182.1 ANXA4
    NM001153.2 ANXA4
    NM001975.1 ENO2
    NM006435.1 IFITM2
    NM001387.1 DPYSL3
    BG398414 RPA1
    NM004039.1 ANXA2
    NM005534.1 IFNGR2
    AL136877.1 SMC4L1
    NM014876.1 KIAA0063
    NM024830.1 FLJ12443
    NM005505.1 SCARB1
    NM003025.1 SH3GL1
    NM013285.1 HUMAUANTIG
    NM005720.1 ARPC1B
    AW157070 EGFR
    NM002835.1 PTPN12
    NM004428.1 EFNA1
    AW006290 SUDD
    NM014791.1 MELK
    NM014882.1 KIAA0053
    NM003864.1 SAP30
    NM001558.1 IL10RA
    NM003264.1 TLR2
    NM014221.1 MTCP1
    AV756141 CSF2RB
    AI123251 LCP2
    NM006433.2 GNLY
    NM000861.2 HRH1
    NM001870.1 CPA3
    NM003586.1 DOC2A
    NM004271.1 MD-1
    NM014932.1 NLGN1
    NM014947.1 KIAA1041
    NM000647.2 CCR2
    NM002562.1 P2RX7
    NM006058.1 TNIP1
    NM013447.1 EMR2
    NM013416.1 NCF4
    NM001776.1 ENTPD1
    NM020037.1 ABCC3
    NM006135.1 CAPZA1
    NM007036.2 ESM1
    AF034607.1 CLIC1
    BC000915.1 PDLIM1
    AL162068.1 NAP1L1
    NM006947.1 SRP72
    L12387.1 SRI
    AF141349.1 N/A
    AF263293.1 SH3GLB1
    BC000389.1 TM4SF7
    AF007162.1 CRYAB
    D38616.1 PHKA2
    AV717590 ENTPD1
    U87967.1 ENTPD1
    H23979 MOX2
    AF063591.1 MOX2
    BC005254.1 CLECSF2
    BC000893.1 H2BFT
    L22431.1 VLDLR
    AI741056 SELPLG
    AF084462.1 RIT1
    U62027.1 C3AR1
    M87507.1 CASP1
    J04132.1 CD3Z
    M31159.1 IGFBP3
    AF257318.1 SH3GLB1
    BC001388.1 ANXA2
    AF130095.1 FN1
    AF022375.1 VEGF
    AA807529 MCM5
    AK026737.1 FN1
    X14355.1 N/A
    AK025608.1 KIAA0930
    AF183421.1 RAB31
    NM002695.1 POLR2E
    AF288391.1 C1orf24
    NM003730.2 RNASE6PL
    NM016359.1 ANKT
    NM014164.2 FXYD5
    NM022736.1 FLJ14153
    NM021158.1 C20orf97
    NM017792.1 FLJ20373
    NM020142.1 LOC56901
    NM016448.1 RAMP
    NM005767.1 P2Y5
    NM020169.1 LXN
    NM022834.1 FLJ22215
    NM018460.1 BM046
    NM024629.1 FLJ23468
    NM018641.1 C4S-2
    NM018295.1 FLJ11000
    NM024576.1 FLJ21079
    NM016582.1 PHT2
    NM003116.1 SPAG4
    NM018454.1 ANKT
    NM018099.1 FLJ10462
    NM007072.1 HHLA2
    NM022445.1 TPK1
    AW173623 TDE1
    AB044088.1 BHLHB3
    AF043244.1 NOL3
    AF133207.1 H11
    AF313468.1 CLECSF12
    AA191576 NPM1
    AI765383 KIAA1466
    BC003654.1 SLC27A3
    W60806 N/A
    AI335263 NETO2
    AI378406 EGLN3
    BC005400.1 FKSG14
    AI761520 CENTA2
    BC000771.1 TPM3
    BC000190.1 HSPC216
    BC002776.1 SEMA5B
    AF132203.1 SCD
    BC006107.1 ARHGAP9
    AK024263.1 N/A
    AK024846.1 SET7
    BE878463 N/A
    AW304786 PTR4
    AI769269 N/A
    AI935334 N/A
    BF437747 SAMHD1
    AW300953 N/A
    H37811 N/A
    AA603344 SAMHD1
    AA742310 N/A
    AI248208 FLJ25804
    AI962367 ECGF1
    NM002053.1 GBP1
    NM000089.1 COL1A2
    NM021105.1 PLSCR1
    NM002467.1 MYC
    NM001284.1 AP3S1
    AI825926 PLSCR1
    NM014736.1 KIAA0101
    AF161461.1 LEPROTL1
    NM014873.1 KIAA0205
    AI005043 N/A
    NM000416.1 IFNGR1
    NM004172.1 SLC1A3
    NM004207.1 SLC16A3
    AI761561 HK2
    Y09216.1 N/A
    NM002922.1 RGS1
    NM005990.1 STK10
    NM014863.1 GALNAC4S-6ST
    NM014737.1 RASSF2
    NM000418.1 IL4R
    BC000658.1 STC2
    NM003751.1 EIF3S9
    NM002339.1 LSP1
    NM004604.1 STX4A
    NM006404.1 PROCR
    AF275945.1 EVA1
    NM004221.1 NK4
    NM004556.1 NFKBIE
    NM004688.1 NM
    NM003332.1 TYROBP
    NM015136.1 STAB1
    NM006019.1 TCIRG1
    NM004877.1 GMFG
    NM002317.1 LOX
    NM025201.1 PP1628
    NM014800.1 ELMO1
    L41944.1 IFNAR2
    NM007268.1 Z39IG
    NM006994.2 BTN3A3
    AF091352.1 VEGF
    AB035482.1 ICB-1
    Z24727.1 TPM1
    M19267.1 TPM1
    U13700.1 CASP1
    M27281.1 VEGF
    BC005838.1 N/A
    BC005858.1 FN1
    BC005926.1 EVI2B
    BE513104 YARS
    AU147399 CAV1
    AK023154.1 HN1L
    AK021757.1 KIAA0648
    H95344 VEGF
    AB023231.1 FNBP4
    AL523076 N/A
    NM030666.1 SERPINB1
    AB018289.1 KIAA0746
    AW043713 SULF1
    BE880591 EP400
    AU158495 NOTCH2
    BE965029 N/A
    AL564683 CEBPB
    AA349595 RAB6IP1
    AI809341 PTPRC
    AW205215 KIAA0286
    BE349017 HA-1
    AF070592.1 HSKM-B
    AI769685 CARS
    AI935123 LOC113146
    BG255188 N/A
    AI088622 PRKCDBP
    BE222709 N/A
    AW007573 DKFZP586L151
    BG332462 N/A
    AI862658 FEM1C
    AI934469 KIAA0779
    AB018345.1 KIAA0802
    W87466 LOC92689
    BE908217 ANXA2
    NM005615.1 RNASE6
    BE300252 K-ALPHA-1
    BF740152 MYO1F
    AV711904 LYZ
    AW072388 N/A
    AW190316 SHMT2
    NM005412.1 SHMT2
    NM006417.1 IFI44
    AL008730 C6orf4
    L16895 LOC114990
    Z21533.1 HHEX
    AK022955.1 DKFZp762L0311
    BF001267 N/A
    AL558987 N/A
    AA577672 LOC151636
    BE620734 ZAK
    AI937446 N/A
    H99792 N/A
    BE966748 N/A
    AI659418 MGC21854
    AI990891 DKFZp761K2222
    AA827892 N/A
    AL135264 N/A
    AI375753 N/A
    AA573502 TAP2
    BG387557 CASP2
    AA554833 MAP1B
    AK026764.1 N/A
    AU146532 PDK1
    BE348597 N/A
    AL577758 LOC133957
    AI133452 FGG
    AU157224 N/A
    AI742057 N/A
    BE500942 N/A
    N25631 RFXANK
    AU145366 N/A
    AW270037 KIAA0779
    BF526978 N/A
    AW182575 N/A
    BF339831 MGC13114
    AI056992 N/A
    BE222668 N/A
    BG165011 N/A
    AI188445 MGC14289
    BE551416 HAK
    AI972498 a1/3GTP
    AW662189 N/A
    AA142842 N/A
    BF939473 N/A
    AI681260 N/A
    AA551090 AP1S2
    AA045175 MS4A6A
    W05495 N/A
    AI093231 N/A
    AI565054 N/A
    AL553774 N/A
    AK023470.1 MGC15875
    AL157377 ENPP3
    AL139109 TEX11
    AK025631.1 POLH
    AI873425 N/A
    BF541967 N/A
    AI686890 N/A
    AI936034 ITGA4
    U88964 ISG20
    AJ243797 TREX1
    D29642 KIAA0053
    D87433 STAB1
    AI129310 FLJ21562
  • TABLE 4
    Genes With Down-Regulated Expression
    In stage II Renal Cell Carcinoma
    Genbank ID Gene Symbol
    NM012248.1 SPS2
    NM002300.1 LDHB
    BC000306.1 HADHSC
    NM001640.2 APEH
    NM005875.1 GC20
    NM003365.1 UQCRC1
    BF031714 HYA22
    NM005808.1 HYA22
    AF113129.1 ATP6V1A1
    NM002402.1 MEST
    NM006844.1 ILVBL
    NM004636.1 SEMA3B
    NM002496.1 NDUFS8
    NM006556.1 PMVK
    NM004255.1 COX5A
    NM002225.2 IVD
    NM004524.1 LLGL2
    AI950380 BCL7A
    AB020707.1 WASF3
    NM000481.1 AMT
    NM012317.1 LDOC1
    NM006456.1 STHM
    NM006614.1 CHL1
    NM015393.1 DKFZP564O0823
    AV729634 DNAJC6
    NM002628.1 PFN2
    NM003500.1 ACOX2
    NM002655.1 PLAG1
    NM004393.1 DAG1
    NM003026.1 SH3GL2
    NM002010.1 FGF9
    NM014033.1 DKFZP586A0522
    NM004868.1 GPSN2
    BC000649.1 UQCRFS1
    S69189.1 ACOX1
    AF153330.1 SLC19A2
    AF094518.1 ESRRG
    M55575.1 BCKDHB
    BE044480 MGC32124
    BF382393 N/A
    AV751731 PNKP
    U55984 N/A
    BF059512 DNER
    AK025934.1 Evi1
    AL036088 SEMA6D
    BE964222 FLJ38482
    AW290940 N/A
    AL545998 N/A
    AW274874 N/A
    AI709389 N/A
    BF224092 MGC15854
    AU145805 N/A
    AW079843 MGC33338
    AW138815 N/A
    AW242286 N/A
    AW025023 N/A
    BE672659 N/A
    AB019695.1 TXNRD2
    M61900.1 PTGDS
    BF967998 N/A
    BF967998 N/A
    AL526243 KIAA0446
    NM000532.1 PCCB
    BE042354 LDHB
    AI587323 ATP5A1
    AW195882 ATPW
    H71135 ADH6
    AV659180 ALDOB
    AK027006.1 TNRC9
    AV693216 PLXNB1
    BG398937 N/A
    NM002489.1 NDUFA4
    NM003849.1 SUCLG1
    NM014019.1 HSPC009
    NM024952.1 FLJ20950
    NM014185.1 MOG1
    NM018013.1 FLJ10159
    NM018373.1 SYNJ2BP
    NM014067.2 LRP16
    NM013261.1 PPARGC1
    NM021963.1 NAP1L2
    NM018658.1 KCNJ16
    NM014553.1 LBP-9
    AF112204.1 ATP6V1H
    AU145941 CDC14B
    AF061264.1 MGC4825
    BF941492 FLJ10496
    AI984229 HSPC121
    N71923 FLRT3
    BC005050.1 NICN1
    AF172327.1 N/A
    AF356515.1 HINT2
    BE620739 RHOBTB3
    BF435123 N/A
    AW149498 BTBD6
    AW024437 LOC118491
    AW195353 N/A
    BE044193 N/A
    AI493303 FLJ31709
    AI636080 N/A
    BF509031 ATP6V1G3
    AW242920 N/A
    BF002046 ANGPTL1
    BF130943 N/A
    AW452631 N/A
    AI792937 N/A
    AI810572 N/A
    BG165743 LOC112817
    AW466989 N/A
    R48991 N/A
    BF029215 MSI2
    D21851 LARS2
    Z83838 ARHGAP8
  • TABLE 5
    Genes With Up-Regulated Expression In both
    stage I & stage II Renal Cell Carcinoma
    Genbank ID Gene Symbol
    NM005566.1 LDHA
    NM000291.1 PGK1
    NM001219.2 CALU
    NM002966.1 S100A10
    NM000034.1 ALDOA
    NM002627.1 PFKP
    NM006082.1 K-ALPHA-1
    AI922599 VIM
    NM020474.2 GALNT1
    NM006406.1 PRDX4
    NM015344.1 LEPROTL1
    NM014755.1 TRIP-Br2
    AI796269 NBS1
    NM005783.1 APACD
    BF197655 N/A
    NM001233.1 CAV2
    NM002845.1 PTPRM
    NM014302.1 SEC61G
    U47924 CD4
    NM004106.1 FCER1G
    NM015474.1 SAMHD1
    NM004915.2 ABCG1
    NM002432.1 MNDA
    NM005565.2 LCP2
    NM005531.1 IFI16
    NM005849.1 IGSF6
    NM002189.1 IL15RA
    NM004353.1 SERPINH1
    NM017760.1 FLJ20311
    NM022349.1 MS4A6A
    NM023003.1 TM6SF1
    NM016184.1 CLECSF6
    NM031284.1 DKFZP434B195
    BC002342.1 CORO1C
    AA775177 PTPRE
    AL162070.1 CORO1C
    AF253977.1 MS4A6A
    AF237908.1 MS4A6A
    W03103 DDEF1
    AK022888.1 FENS-1
    AI141784 N/A
    NM014812.1 KIAA0470
    AF208043.1 IFI16
    BC002654.1 TUBB-5
    BC006379.1 K-ALPHA-1
    BC006481.1 K-ALPHA-1
    AF000426.1 LST1
    AF000424.1 LST1
    BG500301 ITGB1
    AL516350 ARPC5
    M27487.1 HLA-DPA1
    M27487.1 HLA-DPA1
    AW517686 ATP2B4
    AL581768 K-ALPHA-1
    AA524505 TSGA
    Z78330 ACTR3
    Z78330 ACTR3
    BG532690 ITGA4
    AW005535 RAP2B
    NM007161.1 LST1
    AK026577.1 ALDOA
    AI091079 SHC1
    AV713720 LST1
    NM021103.1 TMSB10
    NM016337.1 RNB6
    NM013260.1 HCNGP
    NM021199.1 SQRDL
    NM018149.1 FLJ10587
    NM016951.2 CKLF1
    AB033038.1 FLJ10392
    AI184968 C1QG
    AL161725 FLJ00026
    NM018440.1 PAG
    AL553942 FLJ31951
    AI394438 N/A
    T64884 N/A
    T64884 N/A
    AW511319 N/A
    AI640834 RA-GEF-2
    AI655467 N/A
    AL161725 FLJ00026
    T92908 N/A
  • TABLE 6
    Genes With Down-Regulated Expression In Both
    stage I And stage II Renal Cell Carcinoma
    Genbank ID Gene Symbol
    NM004092.2 ECHS1
    NM000270.1 NP
    NM002354.1 TACSTD1
    AF017987.1 SFRP1
    NM003012.2 SFRP1
    NM000666.1 ACY1
    NM000191.1 HMGCL
    NM015254.1 KIF13B
    NM000140.1 FECH
    U75667.1 ARG2
    NM000196.1 HSD11B2
    NM014636.1 RALGPS1A
    NM001441.1 FAAH
    NM005978.2 S100A2
    NM001678.1 ATP1B2
    NM001099.2 ACPP
    NM014731.1 ProSAPiP1
    BF343007 N/A
    NM000035.1 ALDOB
    NM005950.1 MT1G
    NM002371.2 MAL
    NM006984.1 CLDN10
    NM002567.1 PBP
    NM000019.1 ACAT1
    NM001692.1 ATP6V1B1
    X77737.1 N/A
    NM006226.1 PLCL1
    NM000893.1 KNG
    NM000412.2 HRG
    NM001963.2 EGF
    NM003361.1 UMOD
    NM000050.1 ASS
    NM001438.1 ESRRG
    NM020632.1 ATP6V0A4
    AI632015 SLC12A1
    NM000701.1 ATP1A1
    NM031305.1 DKFZP564B1162
    AF130089.1 ALDH6A1
    AK025651.1 N/A
    W45551 MMP24
    W67995 FXC1
    AL136566.1 IBA2
    AF105366.1 SLC12A6
    AF284225.1 DMRT2
    AA191708 N/A
    AL355708.1 N/A
    BE783949 FLJ10101
    AL529672 N/A
    AL568674 MYBBP1A
    AU147564 CLMN
    AK000208.1 N/A
    AB051536.1 FLJ14957
    AI569747 TFDP2
    AK025562.1 N/A
    AI660243 TMPRSS2
    N50413 N/A
    AI347918 N/A
    AL536553 GRP58
    BC000282.1 LOC89894
    BF106962 FAM3B
    AI051248 FLJ32115
    AI928242 N/A
    BG236006 N/A
    AI653107 N/A
    AI824037 FLJ25461
    R61322 N/A
    AW071744 KCNJ10
    BF059276 N/A
    BC002449.1 FLJ13612
    J02639.1 SERPINA5
    BC002571.1 DKFZP564O243
    U03884.1 KCNJ1
    AF173154.1 HYAL1
    AF130103.1 PBP
    AL117618.1 PDHB
    AF063606.1 N/A
    BC005314.1 N/A
    BF686267 PBP
    AI742553 PRKWNK1
    D83782.1 SCAP
    AB029031.1 TBC1D1
    AK025432.1 KIAA0564
    AL117643.1 N/A
    AW772192 N/A
    NM003944.1 SELENBP1
    AL049977.1 CLDN8
    AK023937.1 THEA
    AK025084.1 TNRC9
    X03363.1 ERBB2
    AK026411.1 ALDOB
    NM016026.1 RDH11
    NM016286.1 DCXR
    NM019027.1 FLJ20273
    BG338251 RAB7L1
    NM006113.2 VAV3
    NM018075.1 FLJ10375
    NM013271.1 PCSK1N
    NM017586.1 C9orf7
    NM016321.1 RHCG
    NM025247.1 MGC5601
    BC002449.1 FLJ13612
    AI379517 N/A
    AA058832 MGC33926
    AW274034 N/A
    AI580268 NUDT6
    AI761947 DKFZP564B1162
    AI793201 N/A
    AK025898.1 N/A
    AB046810.1 C20orf23
    AK024204.1 N/A
    BF594722 N/A
    R88990 N/A
    N73742 N/A
    AI697028 FLJ90165
    BF590528 N/A
    AI733359 N/A
    H20179 N/A
    AA991551 MGC14839
    AI758950 SLC26A7
    AA911561 N/A
    AI769774 N/A
    AA669135 N/A
    AW136060 SLC13A2
    AI733593 N/A
    BF739841 N/A
    AA600175 N/A
    BF477980 N/A
    AI934557 N/A
    BE326951 KNG
    AI632567 N/A
    BE300882 N/A
    BE855713 N/A
    AA485440 DBP
    AA915989 FLJ10743
    AA085764 SIGIRR
    • Example 6 Loss of TGF-β Receptor Expression Demonstrated by Gene Array and Real-Time PCR in Renal Cell Carcinoma
  • Expression of type I TGF-β receptor (TBR1), type II TGF-β receptor (TBR2), and type III TGF-β receptor (TBR3) mRNA were compared in normal renal tissue, primary renal cell carcinoma without metastasis, primary lesions of metastatic renal cell carcinoma, and metastatic lesions. A summary of gene array analysis was presented as average signal intensities in FIG. 11A (mean±standard error). The signal intensity for TBR1 (cross-hatched bars) was relatively low, although TBR1 was scored as ‘Present’ in all samples. No significant changes in TBR1 expression were observed. TBR2 (gray bars) was abundantly expressed in normal epithelium and in primary lesions of nonmetastatic renal cell carcinoma. TBR2 was significantly reduced in primary lesions with metastatic disease (P<0.028 by ANOVA). TBR2 was even more reduced in metastatic lesions. TBR3 expression was high in normal epithelium, but was significantly reduced in each of the five primary tumors with nonmetastatic disease (black bars). TBR3 expression was also reduced in primary tumors with metastatic lesions and in metastatic lesions themselves.
  • These expression patterns were confirmed by real-time PCR (Tagman®) in the 10 patients used for gene array analysis. Means and standard errors for individual samples are shown in FIG. 11B. All data were normalized to 18S rRNA and calibrated to target abundance in the paired normal tissues. TBR1 mRNA abundance did not change (cross-hatched bars), consistent with the gene chip data. TBR2 (gray bars) was not reduced in primary tumors without metastases, whereas TBR2 was significantly reduced in primary tumors with metastatic disease and in metastatic lesions. TBR3 was reduced in all tumors (black bars).
  • The investigators have subsequently completed real-time PCR analysis of TBR1, TBR2, and TBR3 expression in 16 primary tumors without metastases (plus paired normal epithelium) and nine samples of primary tumors with metastatic disease, paired metastatic lesions, and paired normal tissue. The data were consistent with those shown for the samples analyzed in FIG. 11A. TBR3 expression was significantly reduced in all tumors; whereas TBR2 expression was reduced in only 1/16 primary tumors without metastatic lesions, but was reduced in primary tumors with metastatic lesions (8/9). These data show that loss of TBR3 is an early event in renal cell carcinoma, strongly suggesting that TBR3 plays a critical role in renal cell carcinoma carcinogenesis.
  • The loss of TBR3 mRNA expression was also correlated with TNM scores (T, histological score; N, lymph node number; M, number of organ metastases) from patient samples (data not shown). TBR3 mRNA expression was suppressed in the earliest stage, stage I, and was found to be suppressed in all tumor stages (I-IV). In addition, loss of TBR2 in the primary tumor is significantly associated with acquisition of the metastatic phenotype and clinically manifests as metastatic progression.
    • Example 7 Attenuation of TGF-β-Mediated Signal Transduction In Human Renal Cell Carcinoma
  • Decreased type III TGF-β receptor (TBR3) mRNA expression in all tumors was associated with failure to detect TBR3 protein by immunohistochemistry (FIG. 12). Type I TGF-β receptor (TBR2) protein was detected in localized tumor (primary, no mets), but was not detectable in primary tumors with metastatic disease or in corresponding metastatic lesions. Type I TGF-β receptor (TBR1) protein was detected in normal tissue and in all tumor samples.
  • The investigators hypothesized that these losses seen in TGF-β receptor expression would manifest as an attenuation of TGF-β mediated signal transduction, and would significantly alter the expression of TGF-β regulated genes. From the gene array data disclosed above, 13 known TGF-3/Smad-regulated genes were down-regulated in renal cell carcinoma (Table 7). Using mRNA from 35 patient-matched samples, the investigators verified loss of expression of three of these genes by comparing matched normal and tumor tissue. Real-time PCR was used to measure the expression of Collagen IV type 6, fibulin-5, and connective-tissue growth factor (CTGF). Collagen IV type 6 (gray bars) is an extracellular matrix protein that plays a critical role in the regulation of membrane integrity and cell signaling. Fibulin-5 is a recently discovered TGF-3-regulated gene, which has tumor suppressor activity. Fibulin-5 is an extracellular matrix protein that is believed to signal through interaction with integrins. CTGF is a secreted protein involved in angiogenesis, skeletogenesis, and wound healing. CTGF enhances TGF-β1 binding to TBR2, and CTGF and TGF-β collaborate to regulate the expression of extracellular matrix proteins during renal fibrosis. As summarized graphically in FIG. 13, all the evaluated TGF-β-regulated genes were down-regulated in early tumor stages, suggesting that renal cell carcinoma undergoes loss of TGF-β responsiveness at an early stage. These data indicate that this loss of TGF-β sensitivity is due, primarily, to loss of type III TGF-β receptor (TBR3) in early tumor development and further loss of sensitivity in metastatic disease is mediated through subsequent loss of type II TGF-β receptor (TBR2).
  • TABLE 7
    Known TGF-β-Regulated Genes Found To Be Down-
    Regulated In Localized Tumors By Gene Array Analysis
    Fold
    GenBank No. Gene Name Attenuation
    S81439 TGFβ-induced early growth factor 2.5
    (TIEG)
    AF093118 Fibulin 5 4.0
    U42408 Ladinin 1 15.4
    U01244 Fibulin 1 4.8
    J05257 Dipeptidase 1 7.7
    D21337 Collagen, type IV, a6 3.6
    X80031 Collagen, type IV, a3 2.4
    M64108 Collagen, type XIV, a1 3.2
    M98399 Collagen, type I receptor 4.2
    L23808 Matrix metallo-proteinase 12 3.7
    M35999 Integrin, b3 2.5
    AI304854 p27Kip1 2.1
    J05581 Mucin 1 6.5
    Data were analysed by a combination of two-dimensional ANOVA, Affymetrix MAS5.0, and hierarchical cluster analysis using Spotfire to identify genes that are down-regulated in local tumors versus that of normal renal cortex tissue.
    • Example 8
  • TGF-β Receptor Expression in Renal Cell Carcinoma Cell Lines
  • Human renal cell carcinoma cell lines were identified that recapitulate the clinical observations of TGF-β receptor biology described above. UMRC6 cells were derived from a clinically localized human renal cell carcinoma (Grossman et al., 1985). As shown in FIG. 14A, UMRC6 cells express type II TGF-β receptor (TBR2) mRNA, but not type III TGF-β receptor (TBR3). Immunohistochemical analysis (FIG. 14B) confirms the presence of TBR2 protein and the absence of TBR3 expression. UMRC3 cells were derived from the primary tumor of a patient with metastatic renal cell carcinoma. This highly aggressive cell line lacks detectable TBR2 and TBR3 mRNA (FIG. 14A) and protein (FIG. 14B).
  • In addition to these relevant laboratory models, normal renal epithelial (NRE) tissue was harvested from nephrectomy specimens and established as primary cultures
  • (Trifillis, 1999). As shown in FIGS. 14A and 14B, these primary cultures of NRE expressed TBR3, TBR2, and TBR1 mRNA and protein in vitro. NRE cells can be grown in culture for 10 passages and were easily isolated and characterized. NRE cells were characterized for cytokeratin expression and tubule-specific gene expression, for example, megalin (data not shown). Thus, there are relevant cell models in which TBR2 and TBR3 expression can be manipulated to examine the impact of TGF-β receptor biology on the carcinogenesis and progression of human renal cell carcinoma in vitro.
    • Example 9
  • TGF-β Activity In Renal Cell Carcinoma Cell Lines
  • It is well known that TGF-β1 inhibits cell proliferation in epithelial cells. The present example demonstrates the effects of TGF-β on renal tumor cell proliferation. DNA content of cells was used as a measure of cell proliferation. Cells were plated at 20,000 cells/well in 12-well plates. Cells were grown in 10% FBS:DMEM:penicillin:streptomycin. The following day, media were exchanged with appropriate treatment added to the media. On day 3 of treatment, cells were analyzed for DNA content using Hoechst reagent. DNA standard was used to correlate DNA content per well. As shown in FIG. 15A (squares), TGF-β1 inhibited the proliferation of normal renal epithelial cells in culture. URMC3 cells expressed neither type II or type III TGF-t3 receptors and, not surprisingly, were resistant to the inhibitory effects of TGF-β on cell proliferation (triangles, FIG. 15A). UMRC6 cells expressed type II but not type III TGF-β receptors, and were partially resistant to TGF-β1 (circles, FIG. 15A).
  • TGF-β transcriptional activity was also measured in the above cell models using transient transfection of the 3TP/lux reporter, which contains an AP-1/Smad3 response element from the PAI-1 promoter. This luciferase reporter construct demonstrates increased transcriptional activity in response to exogenous TGF-β-mediated signal transduction. 3TP/lux was transiently transfected along with SV/renilla luciferase (Promega) into cells using fugene (Roche) as the transfection agent. Cells were treated with or without TGF-β1 24 h after transfection and luciferase activity (Promega Luciferase Assay system and Lumat luminometer) was determined 24 h after TGF-β treatment. Firefly luciferase activity was normalized using the ratio of firefly luciferase/renilla luciferase. As shown in FIG. 15B, normal renal epithelial cells were highly responsive to 2 ng/ml (80 pM) of TGF-β1. UMRC6 cells demonstrated significantly less luciferase activity in response to TGF-β1, and UMRC3 cells were entirely unresponsive.
    • Example 10
  • Recapitulation of TGF-β Signaling Through Reintroduction of TGF-b Receptor Expression into Renal Cell Carcinoma
  • To test whether reintroduction of TGF-β receptor expression would result in re-establishment of TGF-β signal transduction and reacquisition of TGF-β cellular sensitivity, UMRC3 cells were engineered to express stably either type II TGF-β receptor (+TBR2) alone or type II plus type III TGF-β eceptor (+TBR2+TBR3).
  • Plasmid construction and transfection were described as follows. The complete coding sequences for human type II TGF-β receptor (TBR2) was cloned into the EcoRI/XbaI site of pcDNA3/FLAG. The expression vector was stably transfected into UMRC3 cells using fugene as DNA carrier and genticin as selection antibiotic (Sigma, 1 mg/ml). Ten clones (UMRC3/TBR2) were selected and verified for TBR 2 mRNA and protein expression such as Western analysis using the FLAG antibody (data not shown). From these cell clones, one was to be selected that had equivalent protein expression of TBR2 to that of normal renal epithelial (NRE) and UMRC6 cells.
  • The type III TGF-β receptor (TBR3) coding sequence was PCR amplified from a plasmid expressing wild-type TBR3 in pSV7d (a gift from Dr C-H Heldin). TBR3 was then cloned into the EcoRI site of pcDNA4/TO/myc-His® (InVitrogen) in the sense and antisense (negative control) orientation. The orientation and sequence of TBR3 was verified. The antisense TBR3 (As TBR3) vector was used as a control. TBR3/pcDNA4/TO/myc-His and As TBR3/pcDNA4/TO/myc-His vectors were stably transfected into UMRC3/TBR2 cells. A clone was selected that demonstrated an equivalent expression of TBR3 mRNA to that of normal renal epithelial cells. As a control for UMRC3+TBR2 and UMRC3+TBR2+TBR3, wild-type UMRC3 were stably transfected with both pcDNA/FLAG and pcDNA4/TO/myc-His vectors.
  • As shown in FIGS. 16A-16B, stable transfection of type II TGF-β receptor (TBR2) alone or type II plus type III TGF-β receptor (TBR2+TBR3) resulted in detectable levels of mRNA for each receptor on RT-PCR analysis. On examining the in vitro growth kinetics of these re-engineered cells, it was noted that reintroduction of TBR2 resulted in a twofold reduction in cell proliferation and reintroduction of both TBR2 and TBR3 resulted in a fourfold reduction in cell proliferation with the addition of exogenous TGF-3.
  • The investigators then examined TGF-13-mediated transcriptional activity as a consequence of TGF-β receptor re-expression. As shown in FIG. 16C, reintroduction of TBR2 partially restored transcriptional responsiveness, as evidenced by a 5.6-fold increase in 3TP/lux activity after addition of TGF-β1. Reintroduction of both TBR2 and TBR3 into UMRC3 cells resulted in 17.5-fold increase in 3TP/lux activity after addition of TGF-β1.
  • To demonstrate reestablishment of TGF-β-regulated gene expression, collagen IV type 6 mRNA expression was examined by real-time PCR in these re-engineered cell lines in the presence of TGF-β1. As shown in FIG. 16D, reexpression of TBR2 in UMRC3 cells results in a sevenfold increase in collagen IV type 6 mRNA levels over that of UMRC3 controls, while reintroduction of both TBR2 and TBR3 enhanced collagen IV type 6 mRNA expression 11-fold. These data are consistent with a number of published reports that indicate expression of TBR3 is essential for full TGF-β responsiveness.
  • UMRC3 cells have been shown to be tumorigenic in athymic nude mice (Grossman et al., 1985). Anchorage independent growth in soft agar is a well-established in vitro correlate of in vivo tumorigenicity. Colonies formation in soft agar was determined as follows. UMRC3 (pcDNA/FLAG and pcDNA4/T0/myc-His empty vectors), UMRC3+TBR2, or UMRC3+TBR2+TBR3 cells were plated at 1000 cells/60 mm dish in an agarose/FBS/media sandwich in the presence of 2 ng/ml TGF-β. No selection antibodies were added to the agarose media mixture. The cells were incubated for 45 days to insure that no colony formation would occur. Cells were then stained with 0.005% Crystal Violet, photographed, and assessed for number and size of colonies.
  • As shown in FIG. 16E, UMRC3 cells demonstrated anchorage independent growth in soft agar. Reintroduction of TBR2 into UMRC3 cells significantly decreased the number and size of colonies that formed in soft agar. Reintroduction of both TBR2 and TBR3 completely abrogated the ability of UMRC3 cells to form colonies in soft agar, even after 45 days in culture. These data demonstrate that reintroduction of TBR2 resensitizes UMRC3 cells to the effects of exogenous TGF-β through reacquisition of TGF-β signal transduction. More interestingly, however, reintroduction of TBR3 in the presence of TBR2 into UMRC3 cells significantly enhanced TGF-β-regulated gene transcription, growth inhibition, and loss of anchorage-independent growth over that seen with reintroduction of TBR2 alone. These data clearly show that renal cell carcinoma cells are TGF-β resistant. Loss of TBR3 expression occurs early and appears to be associated with a relatively less aggressive state that is partially TGF-β responsive. Loss of TBR2 results in frank TGF-β resistance and is associated with acquisition of a more aggressive phenotype.
  • FIGS. 17-18 demonstrate that re-expression of type II or type III TGF-β receptor in the highly metastatic human renal cell carcinoma cell line UMRC3 inhibited cell proliferation in cell culture and tumor growth in a nude mouse model. The TGF-β receptors were either re-expressed in a stable vector system or as an adenoviral vector. For clinical purposes, it would be envisioned to treat patients with an adenovirus expressing one or both of the TGF-β receptors to block tumor growth or cause tumor regression.
    • Example 11 Stepwise Sequential Loss of Type III and Type II TGF-β Receptor Expression in Renal Cell Carcinoma
  • With genomic profiling in human renal cell carcinoma, the data presented above demonstrated a stepwise sequential loss of type III and type II TGF-β receptor expression in association with renal cell carcinogenesis and progression. These findings were confirmed by both immunohistochemistry and real-time PCR in patient-matched tissue samples. This clinical observation was brought to the laboratory to identify relevant in vitro models. Using these models, it was demonstrated that loss of type III TGF-β receptor expression resulted in incremental desensitization to TGF-β and attenuation of TGF-β signaling. Subsequent loss of type II TGF-β receptor resulted in complete loss of TGF-β sensitivity. With in vitro modulation of TGF-β receptor expression, it was demonstrated that reconstitution of the TGF-β signaling pathway resulted in significant growth inhibition and loss of the aggressive phenotype.
  • These experiments are unique in that clinically relevant observations, which are derived from the evaluation of gene expression in normal renal cortical tissue, localized renal cell carcinoma and metastatic renal cell carcinoma, were brought to the laboratory for validation and experimental manipulation in relevant in vitro models. Other investigators have examined human renal cell carcinoma cell lines and identified alterations in the expression of TGF-β signaling pathway intermediaries, but those observations have not been validated in the clinical biology of renal cell carcinoma. To the investigators' knowledge, few studies have methodically examined the expression of all three TGF-β receptors in patient samples at the protein and mRNA level in an effort to correlate TGF-β receptor expression to disease-specific states of renal cell carcinoma (i.e. localized versus metastatic tumor). A major strength of the present study is that the investigators recognized distinct disease states in renal cell carcinoma, associated them with specific alterations in the TGF-β signaling pathway, and then validated and manipulated the clinical observations in the laboratory.
  • Although the mechanisms are not well understood, it is clear that TGF-β regulates a large number of diverse biological functions, including cell proliferation, differentiation, cell adhesion, apoptosis, extracellular matrix production, immune regulation, neuroprotection, and early embryonic development. In epithelial cells, the effect of TGF-β is generally to inhibit proliferation, promote cellular differentiation, and regulate interactions with the extracellular matrix. As a direct consequence, aberrations in TGF-β signaling can have a dramatic impact on cellular processes that are critically associated with neoplastic and malignant transformation. Given the well-documented observation that the end result of TGF-β signaling is largely growth inhibitory, it makes intuitive sense that cancer cell would develop mechanisms to escape TGF-β sensitivity. To date, these mechanisms have not been elucidated in human renal cell carcinoma.
  • Based on the data presented above, the investigators hypothesize that this escape from the growth-inhibitory effects of TGF-β is mediated through the stepwise sequential loss of type III and type II TGF-β receptor expression. To the investigators' knowledge, no one has linked sequential loss of these two types of receptors to carcinogenesis and metastatic progression in oncology. This is the first time that stepwise loss of a single transduction pathway has been associated with important biologic sequelae in a human cancer.
  • Results presented in the present invention demonstrate that loss of type III TGF-β receptor expression is an early event in renal cell carcinoma biology and that this loss has important sequelae with regard to renal cell carcinoma carcinogenesis and progression. All clinical samples of localized renal cell carcinoma demonstrated loss of type III TGF-β receptor, but had normal expression of type I and type II TGF-β receptors. Replication of this clinical observation in in vitro models demonstrated significant loss of TGF-β sensitivity, manifest as a significant reduction in the growth inhibitory effects of TGF-β1 and significantly reduced TGF-β-mediated transcription. Interestingly, cell lines derived from localized RCC retained type II TGF-β receptor expression and therefore, still demonstrated sensitivity, albeit reduced, to TGF-β. Only with metastatic progression and loss of type II TGF-β receptor expression does the cell become completely resistant to the effects of TGF-β. The investigators hypothesize that this retained, but attenuated, TGF-β signaling seen in local tumors must convey some as yet unrecognized biologic benefit for local tumors that is no longer required, and therefore discarded, with metastatic progression. In fact, this loss of type II TGF-β receptor expression may be an absolute integral component in the cascade of intracellular events that lead to the development of metastatic potential. In keeping with this hypothesis, it has been shown that loss of type I TGF-b receptor expression was one of 40 integral alterations of gene expression to predict for poor prognosis of patients diagnosed with renal cell carcinoma.
  • In summary, the above results demonstrate a clear link between loss of type III TGF-β receptor expression to a human disease state. Reduced type III TGF-β receptor (TBR3) expression has been reported in human breast tumor cell lines, suggesting that loss of TBR3 expression may be a more ubiquitous phenomena in carcinogenesis, rather than an isolated finding in human RCC biology. The fact that the investigators found down-regulation of TBR3 in every renal cell carcinoma specimen studied to date (35 patients) and that re-expression of TBR3 (in the presence of re-expressed TBR2) completely abolish growth on soft agar suggests an important role for TBR3 in normal renal epithelial homeostasis that must be abrogated for renal cell carcinogenesis and progression to occur. Little attention has been given to TBR3 in normal cell biology or the changes in expression that occur with carcinogenesis and progression. Observations from the present invention would suggest that TBR3 plays an important functional role in signaling and that loss of expression is an important event in the acquisition of the tumorigenic and metastatic phenotype
    • Example 12 Genomic Profiling for Stage I Papillary Renal Cell Carcinoma and Benign Oncocytoma
  • FIG. 19 shows hierarchical clustering of genes over-expressed or down-regulated (with at least 2 fold differences) in stage I papillary renal cell carcinoma verses normal renal cortex. Genes over-expressed and down-regulated in stage I papillary renal cell carcinoma are listed in Table 8 and Table 9 respectively. FIG. 20 shows hierarchical clustering of genes over-expressed or down-regulated (with at least 2 fold differences) in benign oncocytoma verses normal renal cortex. Genes over-expressed and down-regulated in benign oncocytoma are listed in Table 10 and Table 11 respectively. FIG. 21 shows venn analysis of gene distribution among stage I renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma. Genes with at least 2-fold differences in expression were filtered at 95% confidence level (CL) in the following 3 t-tests: stage I RCC vs. normal; oncocytoma vs. normal; and stage I papillary renal cell carcinoma vs. normal. Six hundred twenty five genes were present only in stage I RCC (95% CL), 136 genes were present only in oncocytoma (95% CL), 344 genes were present only in stage I papillary renal cell carcinoma (95% CL), and 60 genes were common to stage I RCC, oncocytoma and stage I papillary renal cell carcinoma. FIG. 22 shows venn analysis of gene distribution among stage II renal cell carcinoma (RCC), oncocytoma and stage I papillary renal cell carcinoma. Genes with at least 2-fold differences in expression were filtered at 95% confidence level (CL) in the following 3 t-tests: stage II RCC vs. normal; oncocytoma vs. normal; and stage I papillary renal cell carcinoma vs. normal. One thousand and five genes were present only in stage II RCC (95% CL), 152 genes were present only in oncocytoma (95% CL), 334 genes were present only in stage I papillary renal cell carcinoma (95% CL), and 43 genes were common to stage II RCC, oncocytoma and stage I papillary renal cell carcinoma.
  • TABLE 8
    Genes With Up-Regulated Expression In
    stage I Papillary Renal Cell Carcinoma
    Genbank ID Gene Symbol
    NM_003505 FZD1
    AL035683 B4GALT5
    R56118 N/A
    NM_014575 SCHIP1
    AI694320 ZNF533
    BC031322 N/A
    BF346665 N/A
    BC004283 LOC283639
    AF302786 GNPTAG
    AU121975 PAICS
    NM_016315 GULP1
    AL541302 SERPINE2
    BG391217 C9orf80
    NM_000700 ANXA1
    N30188 N/A
    NM_003651 CSDA
    AI830227 FLII
    U20350 CX3CR1
    NM_005692 ABCF2
    U34074 AKAP1
    AB056106 TARSH
    AU151483 CDH6
    BC026260 TTC3
    AL133001 SULF2
    NM_003358 UGCG
    NM_001282 AP2B1
    AF322067 RAB34
    NM_001540 HSPB1
    N58363 STATIP1
    AF072872 FZD1
    BF247552 SLC38A1
    X69397 CD24
    BC000251 GSK3B
    BF691447 B4GALT5
    AB046817 SYTL2
    AF255647 DKFZP566N034
    BF344237 N/A
    AW242720 LOC143381
    AA115485 MGC3222
    NM_006588 SULT1C2
    NM_000546 TP53
    N92494 JWA
    W74580 MGC3222
    AF131749 PSK-1
    AW026491 CCND2
    NM_012410 PSK-1
    NM_002800 PSMB9
    BF512748 JAK3
    AA404269 PRICKLE1
    M33376 AKR1C1
    AF035321 DNM1
    NM_002862 PYGB
    AF132000 DKFZP564K1964
    L07950 HLA-C
    AF114011 TNFSF13
    BF674052 VMP1
    AI922599 VIM
    AF044773 BANF1
    NM_015925 LISCH7
    NM_001684 ATP2B4
    AI123348 CHST11
    NM_001304 CPD
    NM_006762 LAPTM5
    NM_000211 ITGB2
    AA995910 ALOX5
    NM_018965 TREM2
    AL353715 STMN3
    BC019612 C20orf75
    AF086074 N/A
    NM_005045 RELN
    AI935123 C14orf78
    AL550875 C7orf28B
    L27624 TFPI2
    AL574096 TFPI2
    AA005141 MET
    D86983 D2S448
    AW439242 C6orf68
    AB000221 CCL18
    NM_002121 HLA-DPB1
    U17496 PSMB8
    U05598 AKR1C1
    BF342851 D2S448
    BF311866 PTGFRN
    NM_001449 FHL1
    AA954994 N/A
    Y13710 CCL18
    BG170541 MET
    AB037813 DKFZp762K222
    D28124 NBL1
    NM_021103 TMSB10
    AI949772 N/A
    AC004382 DKFZP434K046
    NM_000248 MITF
    NM_022154 SLC39A8
    AI436813 N/A
    AF007162 CRYAB
    NM_015392 NPDC1
    AL136585 DKFZp761A132
    AB040120 SLC39A8
    NM_138473 SP1
    AU144387 182-FIP
    NM_022763 FAD104
    AI093231 APBB1IP
    NM_000235 LIPA
    AI817079 EXOC7
    NM_004385 CSPG2
    NM_024801 TARSH
    BF218922 CSPG2
    BF590263 CSPG2
    NM_001233 CAV2
    AB020690 PNMA2
    AW188198 TNFAIP6
    NM_007115 TNFAIP6
    AI742838 DOCK11
    AW117264 N/A
    AF016266 TNFRSF10B
    NM_013952 PAX8
    AA771779 ZFP90
    W72333 FLJ21657
    H23979 MOX2
    BG542521 PPM2C
    AF063591 MOX2
    BF247383 BMPR2
    NM_005114 HS3ST1
    BE466145 N/A
    BC005352 TNFAIP8
    AC002045 LOC339047
    BC040558 D2LIC
    U13699 CASP1
    NM_002718 PPP2R3A
    BF476502 MPPE1
    BC034275 LOC253982
    AF279145 ANTXR1
    AV724216 NDRG4
    BG165613 N/A
    NM_018205 LRRC20
    NM_022083 C1orf24
    NM_006169 NNMT
    AF141347 TUBA3
    NM_000064 C3
    AV710838 BCDO2
    AI417917 EHD2
    AI681260 LILRB1
    NM_000389 CDKN1A
    AF288391 C1orf24
    NM_002627 PFKP
    NM_001975 ENO2
    NM_030786 SYNCOILIN
    NM_006169 NNMT
    AI417917 EHD2
    NM_006868 RAB31
    L03203 PMP22
    AF199015 VIL2
    AI873273 SLC16A6
    NM_017821 RHBDL2
    BF740152 MYO1F
    AA954994 N/A
    AI458735 MGC26717
    NM_003254 TIMP1
    AI688631 N/A
    AK026037 N/A
    BG327863 CD24
    NM_016008 D2LIC
    AI394438 LOC253981
    AA947051 D2LIC
    AI819043 N/A
    AI378044 UGCG
    NM_024576 OGFRL1
    M76477 GM2A
    NM_002214 ITGB8
    AI879381 ADCK2
    NM_000152 GAA
    H15129 MEIS4
    L42024 HLA-C
    NM_002178 IGFBP6
    AI761561 HK2
    AA722799 DCBLD2
    NM_003255 TIMP2
    NM_000107 DDB2
    AV699565 CTSC
    NM_000861 HRH1
  • TABLE 9
    Genes With Down-Regulated Expression In
    stage I Papillary Renal Cell Carcinoma
    Genbank ID Gene Symbol
    AF232217 N/A
    AI823572 MGC45438
    AU154994 SLC13A3
    AW979271 N/A
    AF064103 CDC14A
    AI524125 PCDH9
    AI733474 GPR155
    AI767756 HS6ST2
    NM_000412 HRG
    NM_021614 KCNN2
    M13149 HRG
    H17038 N/A
    NM_002010 FGF9
    AI635774 EMCN
    AW007532 IGFBP5
    NM_004070 CLCNKA
    NM_014621 HOXD4
    AI733593 N/A
    NM_020632 ATP6V0A4
    AI697028 FLJ90165
    AA897516 PTGER4
    NM_024307 MGC4171
    J02639 SERPINA5
    NM_000085 CLCNKB
    AA058832 MGC33926
    BF059276 N/A
    BC043647 LOC284578
    AL161958 THY1
    AL121845 KIAA1847
    AY079172 ATP6V0D2
    AA928708 CYP8B1
    H71135 ADH6
    NM_000102 CYP17A1
    Z92546 SUSD2
    AL558479 THY1
    BC005314 ALDOB
    NM_173591 FLJ90579
    BF510426 N/A
    AF331844 SOST
    X77737 SLC4A1
    NM_004392 DACH1
    BC001077 LOC87769
    AA218868 THY1
    BF478120 RECQL5
    BC041158 CYP4A11
    AI623321 MTP
    AI796189 PAH
    NM_021161 KCNK10
    NM_000163 GHR
    AL136880 ESPN
    NM_024426 WT1
    M61900 PTGDS
    AW963951 SIAT7C
    AW340588 MAN1C1
    AI263078 SLC23A3
    BF130943 PPAPDC1
    AI732596 N/A
    AA603467 ZNF503
    R41565 N/A
    AI951185 NR2F1
    NM_002609 PDGFRB
    NM_006984 CLDN10
    BG413612 N/A
    D64137 CDKN1C
    AK026344 PEPP2
    AI670852 PTPRB
    AI693153 GABRB3
    NM_001393 ECM2
    N93191 PR1
    BC005090 AGMAT
    NM_000717 CA4
    D38300 PTGER3
    AI650260 N/A
    BC024226 IFRG15
    BC006294 DHRS10
    NM_003039 SLC2A5
    AI675836 SORCS1
    NM_005276 GPD1
    NM_014298 QPRT
    M10943 MT2A
    NM_005952 MT1X
    NM_002450 MT1X
    NM_002910 RENBP
    BF246115 MT1F
    AF078844 MT1F
    AF170911 SLC23A1
    AF333388 MT1H
    NM_003500 ACOX2
    AA995925 N/A
    NM_001218 CA12
    BF432333 FLJ31196
    NM_001385 DPYS
    NM_003052 SLC34A1
    NM_000778 CYP4A11
    AL136551 SESN2
    NM_000792 DIO1
    NM_016725 FOLR1
    NM_019101 APOM
    NM_014270 SLC7A9
    AF124373 SLC22A6
    NM_016327 UPB1
    NM_024734 CLMN
    NM_016527 HAO2
    NM_003645 SLC27A2
    AB051536 FLJ14957
    NM_025149 FLJ20920
    BC005939 PTGDS
    AL574184 HPGD
    NM_000161 GCH1
    H57166 N/A
    NM_000597 IGFBP2
    NM_000790 DDC
    NM_004668 MGAM
    NM_021027 UGT1A6
    AF348078 GPR91
    NM_016347 NAT8
    AF338650 PDZK3
    BE221817 CNTN3
    NM_004476 FOLH1
    NM_004615 TM4SF2
    NM_023940 RASL11B
    AI742872 SLC2A12
    BC001196 HS6ST1
    AW195353 TFCP2L1
    NM_003122 SPINK1
    NM_144707 PROM2
    AI653981 L1CAM
    AI796169 GATA3
    M96789 GJA4
    N74607 AQP3
    NM_014059 RGC32
    AI572079 SNAI2
    AI056877 N/A
    NM_006206 PDGFRA
    AW771314 MGC35434
    NM_016955 SLA/LP
    AI569804 LOC375295
    NM_001584 C11orf8
    BG261252 EVI1
    NM_006226 PLCL1
    NM_001172 ARG2
    AL050264 TU3A
    BC003070 GATA3
    AL120332 MGC20785
    NM_000459 TEK
    AW242836 LOC120224
    AI926697 Gup1
    NM_000486 AQP2
    AI870306 IRX1
    AW264204 CLDN11
    BF431989 THRB
    AI459140 N/A
    NM_001864 COX7A1
    AI471866 SLC7A13
    AI653107 NRK
    NM_004466 GPC5
    BF195936 KRT18L1
    NM_022454 SOX17
    AW299531 HOXD10
    AL137716 AQP6
    AI332407 SFRP1
    AL565812 PTN
    AI452457 LOC199920
    AI281593 DCN
    M21692 ADH1B
    AI660243 TMPRSS2
    AI754423 FLJ38507
    AA759244 FXYD4
    U75667 ARG2
    NM_000930 PLAT
    AF083105 SOX13
    NM_013231 FLRT2
    BI825302 PR1
    NM_003012 SFRP1
    AF138300 DCN
    AU155612 N/A
    BG435302 EBF
    NM_005978 S100A2
    NM_000900 MGP
    AK026748 DKFZP761M1511
    J03208 DBT
    NM_002345 LUM
    NM_006623 PHGDH
    AF063606 my048
    NM_001647 APOD
    AI935541 N/A
    NM_005558 LAD1
    AW138125 PRODH2
    NM_003877 SOCS2
    AI768894 CGN
    AW772192 N/A
    AF094518 ESRRG
    T40942 ANGPTL3
    NM_001146 ANGPT1
    AI242023 N/A
    BF970431 N/A
    NM_005670 EPM2A
    AW071744 KCNJ10
    AI928242 TFCP2L1
    AI769774 LOC155006
    AW274034 USP2
    NM_004633 IL1R2
    NM_003289 TPM2
    BF512388 C10orf58
    BC005830 ANXA9
    NM_000362 TIMP3
    NM_001438 ESRRG
    AU146204 ENPP6
    AA775681 FLJ23091
    AI393205 ACY-3
    AF017987 SFRP1
    NM_005951 MT1H
    NM_005950 MT1G
    NM_021805 SIGIRR
    AA557324 CYP4X1
    BF528646 DKFZP564I1171
    AW340112 LOC401022
    R73554 IGFBP5
    AI826437 N/A
    AV720650 KIAA0888
    AA780067 HS3ST3B1
    NM_000640 IL13RA2
    AI806338 TBX3
    NM_003155 STC1
    AA931562 N/A
    AI694325 N/A
    AF205940 EMCN
    NM_001290 LDB2
    NM_016242 EMCN
    AW014927 CALB1
    AI758950 SLC26A7
    AK024256 KIAA1573
    BF726212 ANK2
    AI985987 SCNN1G
    AW242408 UPP2
    NM_000860 HPGD
    BF447963 KIAA0962
    BF941499 GPR116
    AW242409 N/A
    BF509031 ATP6V1G3
    NM_000934 SERPINF2
    BF248364 AF15Q14
    AL534095 FLJ23091
    NM_004929 CALB1
    AI222435 N/A
    NM_005397 PODXL
    AI090268 N/A
    AI300520 STC1
    BC006236 MGC11324
    NM_024609 NES
    NM_002591 PCK1
    NM_005410 SEPP1
    AB020630 PPP1R16B
    AF022375 VEGF
    NM_016246 DHRS10
    AA873542 SLC6A19
    U95090 PRODH2
    D26054 FBP1
    AI732994 MGC13034
    NM_000151 G6PC
    AK025651 PNAS-4
    AF161441 N/A
    AF161454 APOM
    NM_022129 MAWBP
    AI733515 MGC52019
    NM_001443 FABP1
    AI433463 MME
    AL049313 N/A
    BF195998 ALDOB
    NM_022829 SLC13A3
    NM_000035 ALDOB
    NM_007287 MME
    NM_003399 XPNPEP2
    NM_000196 HSD11B2
    BF431313 N/A
    NM_004844 SH3BP5
    NM_003206 TCF21
    AI311917 DPYS
    AA843963 PRLR
    NM_017753 PRG-3
    NM_006633 IQGAP2
    NM_001133 AFM
    T90064 N/A
    BF696216 N/A
    NM_004413 DPEP1
    Z98443 FLJ38736
    NM_018456 EAF2
    AW771563 N/A
    NM_014495 ANGPTL3
    AI074145 KMO
    NM_000896 CYP4F3
    NM_001072 UGT1A6
    AI631993 N/A
    NM_000277 PAH
    M74220 PLG
    AI935789 UMOD
    NM_002472 MYH8
    BC020873 CLCNKA
    NM_000550 TYRP1
    AA806965 BTNL9
    NM_020163 LOC56920
    NM_004490 GRB14
    AA788946 COL12A1
    AW242315 N/A
    AI735586 LOC152573
    R88990 N/A
    NM_003278 TNA
    NM_007180 TREH
    AW173045 TBX2
    U28049 TBX2
    NM_001395 DUSP9
    NM_000336 SCNN1B
    U43604 N/A
    BC029135 N/A
    NM_005414 SKIL
    BQ894022 PDE1A
    NM_013335 GMPPA
    NM_003221 TFAP2B
    BF057634 HOXD8
    AA523172 N/A
    AF319520 ARG99
    NM_002885 RAP1GA1
    NM_003361 UMOD
    NM_000142 FGFR3
    NM_000893 KNG1
    BC029135 N/A
    NM_147174 HS6ST2
    NM_000218 KCNQ1
    U03884 KCNJ1
    X83858 PTGER3
    BF439270 N/A
    AA911235 MST1
    NM_000955 PTGER1
    NM_022844 MYH11
    BC042069 N/A
    NM_005518 HMGCS2
    NM_001963 EGF
    AI632015 SLC12A1
    AF339805 N/A
    BF106962 FAM3B
    NM_005019 PDE1A
    AU146305 PDE1A
    NM_000663 ABAT
    AU119437 LOC144997
    BC036095 DRP2
    R49295 N/A
    AI623202 PRDM16
    AW452355 N/A
    AA563621 HSPB6
    X15217 SKIL
    AK095719 N/A
    AI056187 N/A
    AI668598 N/A
    AI700882 SLC13A3
    NM_000963 PTGS2
    AW051712 N/A
    AL832099 MGC33190
    AK057337 LOC145820
    AW300204 SLC30A8
    NM_005856 RAMP3
    AI458003 CYYR1
    AK026877 N/A
    AI632567 N/A
    U91903 FRZB
    AF352728 FLJ12541
    BM128432 IGFBP5
    NM_003102 SOD3
    BE676272 TACC1
    AI692180 PPFIBP2
    AL544576 LOC92162
    NM_017688 BSPRY
    AU146310 N/A
    AI912976 RASGRF2
    U83508 ANGPT1
    L47125 GPC3
    NM_000663 ABAT
  • TABLE 10
    Genes With Up-Regulated Expression In Benign Oncocytoma
    Genbank ID Gene Symbol
    NM_005114 HS3ST1
    AA650558 GNAS
    BF062244 LIN7A
    NM_030674 SLC38A1
    NM_014766 SCRN1
    BC002471 CPLX1
    AF183421 RAB31
    AK022100 KIAA0256
    BF508244 AKR1C2
    BG772511 N/A
    AB037848 SYT13
    AK055769 N/A
    T58048 N/A
    NM_012105 BACE2
    AA992805 LEF1
    AK026420 DMN
    NM_024812 BAALC
    AI057226 N/A
    AW138767 ELOVL7
    NM_013233 STK39
    AF178532 BACE2
    AI521166 LOC283104
    AA005023 NOD27
    AV725364 GPRC5B
    AW195581 GPSM2
    BG503479 B4GALT6
    BF031829 DSG2
    AW975728 SLC16A7
    NM_022495 C14orf135
    AA703159 N/A
    BF247552 SLC38A1
    NM_001673 ASNS
    NM_024622 FLJ21901
    AI565054 N/A
    AW058459 LOC134285
    NM_001233 CAV2
    BC036550 N/A
    BE464483 N/A
    NM_002512 NME2
    AF178532 BACE2
  • TABLE 11
    Genes With Down-Regulated Expression In Benign Oncocytoma
    Genbank ID Gene Symbol
    BF593625 SYK
    AI310001 FLJ22789
    NM_006206 PDGFRA
    NM_003740 KCNK5
    AW138125 PRODH2
    NM_000336 SCNN1B
    BC005314 ALDOB
    AI796189 PAH
    NM_013363 PCOLCE2
    NM_004466 GPC5
    AI627531 N/A
    U28055 MSTP9
    NM_152759 MGC35140
    AW052159 N/A
    NM_017712 PGPEP1
    AI961231 TOX
    AI767962 BNC2
    AF350881 TRPM6
    AU146418 N/A
    BE875072 N/A
    AI653981 L1CAM
    AI634662 SLC13A3
    NM_000486 AQP2
    AW206292 AQP2
    AI572079 SNAI2
    AI694118 N/A
    NM_000142 FGFR3
    U78168 RAPGEF3
    AI913600 UNQ846
    W93847 MUC15
    NM_004616 TM4SF3
    AI935789 UMOD
    NM_007180 TREH
    AL110152 CD109
    AW051599 N/A
    AI796169 GATA3
    AF017987 SFRP1
    BE550027 DKFZp761N1114
    AA535065 KIAA1847
    NM_003361 UMOD
    AI263078 SLC23A3
    M13149 HRG
    AF278532 NTN4
    AI632015 SLC12A1
    NM_000412 HRG
    NM_000893 KNG1
    BG398937 KNG
    AL049977 CLDN8
    N74607 AQP3
    AW071744 KCNJ10
    AW015506 AQP2
    AI927000 SOSTDC1
    AI471866 SLC7A13
    NM_001099 ACPP
    NM_005074 SLC17A1
    AA995925 N/A
    AF352728 FLJ12541
    BF343007 TFAP2A
    NM_016929 CLIC5
    AA911235 MST1
    AA639753 N/A
    NM_004887 CXCL14
    AW771565 AIM1
    AI264671 N/A
    BF510426 N/A
    AV728958 TLN2
    T90064 N/A
    AA218868 THY1
    NM_003104 SORD
    AJ292204 AGXT2
    AI056359 MAPT
    AL568422 DZIP1
    AF339805 N/A
    NM_000163 GHR
    AI042017 NPL
    AW340457 N/A
    BF431199 DEHAL1
    BF432254 MGC15937
    AI368018 GPD1
    AF144103 CXCL14
    NM_016725 FOLR1
    NM_000050 ASS
    AA693817 N/A
    NM_004929 CALB1
    NM_000592 C4A
    AL574184 HPGD
    AA676742 DMGDH
    AI631993 N/A
    AI566130 AK3
    AW024233 GLYAT
    AA873542 SLC6A19
    AK026966 AK3
    NM_022829 SLC13A3
    NM_005950 MT1G
    AV700405 MGC52019
    AI733515 MGC52019
    NM_000860 HPGD
    U95090 PRODH2
    NM_001385 DPYS
    BG401568 SLC16A9
    NM_000846 GSTA1
    BF195998 ALDOB
    NM_004413 DPEP1
    NM_000151 G6PC
    NM_006744 RBP4
    NM_013410 AK3
    NM_000035 ALDOB
    AK026411 ALDOB
    AL135960 CYP4A11
    M74220 PLG
    NM_001713 BHMT
    AW614558 SLC39A5
    Z92546 SUSD2
    NM_000778 CYP4A11
    NM_000792 DIO1
    AI222435 N/A
    D26054 FBP1
    AW025165 SLC22A8
    NM_007287 MME
    AW274034 USP2
    NM_147174 HS6ST2
    AA074145 PRODH
    AL049176 CHRDL1
    NM_020353 PLSCR4
    NM_024803 TUBAL3
    D16931 ALB
    NM_019076 UGT1A10
    AF138303 DCN
    D13705 CYP4A11
    NM_000587 C7
    R49295 N/A
    NM_000385 AQP1
    AI669229 RARRES1
    U36189 C5orf13
    AL110135 FLJ14753
    AW271605 N/A
    BF358386 N/A
    NM_016270 KLF2
    AA905508 LOC128153
    NM_021630 PDLIM2
    AA915989 TBC1D13
    AL565812 PTN
    AI990790 N/A
    BC041158 CYP4A11
    NM_138474 N/A
    NM_002899 RBP1
    AK024256 KIAA1573
    AW779672 SLC17A1
    NM_021161 KCNK10
    BF196891 TPMT
    AY028896 CARD10
    NM_018456 EAF2
    NM_017806 FLJ20406
    X59065 FGF1
    AI650353 DACH1
    AW771563 N/A
    BF431313 N/A
    NM_000896 CYP4F3
    BC005090 AGMAT
    U24267 ALDH4A1
    AI090268 N/A
    AW014927 CALB1
    AL023553 PIPPIN
    AL049313 N/A
    AK021539 NCAG1
    AI220117 MGST1
    NM_020300 MGST1
    NM_022568 ALDH8A1
    BE874872 FAM20C
    NM_004668 MGAM
    BF033242 CES2
    BC004542 PLXNB2
    NM_000204 F
    NM_004525 LRP2
    AA442149 MAF
    NM_000049 ASPA
    AI830469 TFEC
    NM_003759 SLC4A4
    AF169017 FTCD
    AF170911 SLC23A1
    AA865601 LOC123876
    AA863031 MGC32871
    AW136060 SLC13A2
    NM_003041 SLC5A2
    NM_021924 MUCDHL
    AW299568 N/A
    AI927941 N/A
    AI433463 MME
    AL365347 SLC7A8
    AA502331 PRAP1
    NM_024709 FLJ14146
    AF289024 FTCD
    NM_017614 BHMT2
    NM_016347 NAT8
    NM_000277 PAH
    NM_000316 PTHR1
    NM_001091 ABP1
    NM_000790 DDC
    BF217861 MT1E
    BF447963 KIAA0962
    NM_001081 CUBN
    NM_018484 SLC22A11
    AW192692 N/A
    BF000045 TINAG
    BC005830 ANXA9
    NM_025257 C6orf29
    NM_020973 GBA3
    NM_001977 ENPEP
    AI632692 N/A
    BI825302 PR1
    L12468 ENPEP
    AL571375 SCD4
    AL136858 ZMYND12
    NM_024027 COLEC11
    NM_014934 DZIP1
    BG496631 FBI4
    NM_018265 FLJ10901
    AI770035 UPB1
    AF177272 UGT2B28
    NM_004392 DACH1
    N95363 CDKN1C
    AF261715 FOLH1
    NM_000042 APOH
    NM_001393 ECM2
    R88990 N/A
    AA557324 CYP4X1
    AF116645 ALB
    BC015993 MGC27169
    AL558479 THY1
    NM_000785 CYP27B1
    AW051926 AMN
    AA928708 CYP8B1
    BE407830 KIFC3
    AI431643 RRAS2
    AF001434 EHD1
    BC005894 FMO2
    NM_006798 UGT2A1
    BF217861 MT1E
  • The following references were cited herein:
    • Copland et al., Recent Prog. Horm. Res. 58:25-53 (2003).
    • Copland et al., Oncogene 22:8053-62 (2003).
    • Grossman et al., J. Surg. Oncol. 28:237-244 (1985).
    • Trifillis, Exp. Nephrol. 7:353-359 (1999).

Claims (12)

1.-21. (canceled)
22. A method of detecting a renal cell cancer comprising the steps of:
obtaining one or more biological samples comprising renal tissue or renal cells from an individual;
determining a gene expression level of GATA3 in the sample; and
performing statistical analysis on the expression level of the GATA3 gene as compared to that expressed in normal biological samples comprising renal tissue or renal cells, wherein statistically down-regulated gene expression levels would indicate said individual has papillary or clear cell renal cell cancer.
23. The method of claim 22, further comprising the step of measuring the expression levels of TFCP2L1, wherein down-regulated gene expression levels of TFCP2L1 indicate the individual has papillary or clear cell renal cell cancer.
24. The method of claim 22, further comprising the step of measuring the expression levels of TFAP2B, wherein down-regulated gene expression levels of TFAP2B indicate the individual has papillary or clear cell renal cell cancer.
25. The method of claim 22, further comprising the step of measuring the expression levels of DMRT2, wherein down-regulated gene expression levels of DMRT2 indicate the individual has papillary or clear cell renal cell cancer.
26. The method of claim 22, further comprising the step of measuring the expression levels of TFCP2L1, TFAP2B, and DMRT2, wherein down-regulated gene expression levels of TFCP2L1, TFAP2B, and DMRT2 indicate the individual has papillary or clear cell renal cell cancer.
27. The method of claim 22, wherein the step of determining the gene expression level of the GATA3 gene is by DNA microarray, hierarchical cluster analysis, real-time PCR, RT-PCR, or northern analysis.
28. The method of claim 22, wherein the renal cell cancer is a Stage I, II, II or IV renal cancer.
29. A method of detecting a renal cell cancer comprising the steps of:
obtaining one or more biological samples comprising renal tissue or renal cells from an individual;
determining a gene expression level of GATA3 in the sample and at least one gene selected from TFCP2L1, TFAP2B, and DMRT2; and
performing statistical analysis on the expression level of the GATA3 gene as compared to that expressed in normal biological samples comprising renal tissue or renal cells, wherein statistically down-regulated gene expression levels would indicate said individual has papillary or clear cell renal cell cancer.
30. The method of claim 29, further comprising the step of measuring the expression levels of TFCP2L1, TFAP2B, and DMRT2, wherein down-regulated gene expression levels of TFCP2L1, TFAP2B, and DMRT2 indicate the individual has papillary or clear cell renal cell cancer.
31. The method of claim 29, wherein the step of determining the gene expression level of the GATA3 gene is by DNA microarray, hierarchical cluster analysis, real-time PCR, RT-PCR, or northern analysis.
32. The method of claim 29, wherein the renal cell cancer is a Stage I, II, II or IV renal cancer.
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