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Methods for detecting, diagnosing and treating human renal cell carcinoma

a human renal cell carcinoma and gene expression technology, applied in the field of cancer research, can solve the problems incurable renal cell carcinoma, and largely ineffective existing systemic therapies in affecting disease response or patient survival, so as to improve the survival rate of renal cell carcinoma, cure, or stabilize the disease, and prevent or treat renal cell carcinoma.

Inactive Publication Date: 2005-06-16
LUXON BRUCE A +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] 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.

Problems solved by technology

Renal cell carcinoma (RCC) represents a major health issue.
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 prior art is deficient in understanding the molecular differences between renal cell carcinoma and normal renal epithelium.

Method used

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  • Methods for detecting, diagnosing and treating human renal cell carcinoma
  • Methods for detecting, diagnosing and treating human renal cell carcinoma
  • Methods for detecting, diagnosing and treating human renal cell carcinoma

Examples

Experimental program
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example 1

Tissue Banking

[0054] 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

[0055] 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

[0056] Applied Biosystems' assays-by-design or assays-on-demand 20X assay mix of primers and TaqMan® MGB probes (FAM® dye-labeled) for all target genes and predeveloped 18S rRNA (VIC® 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 an 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.

[0057] 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. ...

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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 [0001] This non-provisional patent application claims benefit of provisional patent application 60 / 502,038, filed Sep. 10, 2003, and 60 / 539,838, filed Jan. 28, 2004, now abandoned.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] 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. [0004] 2. Description of the Related Art [0005] 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 inef...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/535C12Q1/68G01N33/574G06F
CPCC12Q1/6886G01N33/6893C12Q2600/112C12Q2600/106
Inventor LUXON, BRUCE A.COPLAND, JOHN A.WOOD, CHRISTOPHER G.
Owner LUXON BRUCE A
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