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WO2006034354A9 - Methodes pour le diagnostic et le traitement de tumeurs et de carcinomes a cellules renales sporadiques associes a la maladie de von hippel-lindau (vhl), d'autres tumeurs et lesions non tumorales homologues sporadiques de la maladie de vhl qui co-expriment l'epo et le recepteur de l'epo - Google Patents

Methodes pour le diagnostic et le traitement de tumeurs et de carcinomes a cellules renales sporadiques associes a la maladie de von hippel-lindau (vhl), d'autres tumeurs et lesions non tumorales homologues sporadiques de la maladie de vhl qui co-expriment l'epo et le recepteur de l'epo

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Publication number
WO2006034354A9
WO2006034354A9 PCT/US2005/033850 US2005033850W WO2006034354A9 WO 2006034354 A9 WO2006034354 A9 WO 2006034354A9 US 2005033850 W US2005033850 W US 2005033850W WO 2006034354 A9 WO2006034354 A9 WO 2006034354A9
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WIPO (PCT)
Prior art keywords
epo
epor
vhl
sporadic
tumors
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Ceased
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PCT/US2005/033850
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English (en)
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WO2006034354A2 (fr
WO2006034354A3 (fr
Inventor
Zhengping Zhuang
Alexander Vortmeyer
Irina Lubensky
Edward Oldfield
Stefan Frank
Barbara Ikejiri
Tim Vogel
Youn-Soo Lee
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US Department of Health and Human Services
Government of the United States of America
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US Department of Health and Human Services
Government of the United States of America
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Publication of WO2006034354A2 publication Critical patent/WO2006034354A2/fr
Publication of WO2006034354A9 publication Critical patent/WO2006034354A9/fr
Publication of WO2006034354A3 publication Critical patent/WO2006034354A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/746—Erythropoetin

Definitions

  • the invention relates to the diagnosis and treatment of von Hippel-Lindau (VHL) disease associated and sporadic renal cell carcinoma, and other VHL associated and sporadic counterpart tumors.
  • VHL von Hippel-Lindau
  • VHL disease is an autosomal dominant disorder characterized by the development of multiple tumors in specific target organs.
  • Retinal hemangioblastomas (HB), central nervous system (CNS) HB, pancreatic microcystic adenomas and neuroendocrine tumors, pheochromocytomas, and renal cysts and carcinomas are the most frequent lesions in VHL disease (1).
  • Tumor formation is believed to be initiated by a germline mutation of the VHL gene followed by inactivation of the wild-type VHL allele (2, 3).
  • Loss of VHL protein function up-regulates the HIF-I (hypoxia- inducible factor) signaling pathway, which in turn mediates up-regulation of hypoxia inducible genes including EPO and VEGF (4, 5).
  • VHL Historically, the median age of survival is 49 years and the most common cause of death in VHL is from neurologic complications of cerebellar HB, or metastatic renal carcinoma. Approximately 53% die of complications of cerebellar HB and 32% die of metastatic renal cell carcinoma (25).
  • VHL The diagnosis of VHL may be missed by physicians unaware of the typical constellation of findings. Even when diagnosed, relatives of the patient may not be properly screened and genetic counseling may not be offered.
  • the radiologist may play an important role in recognizing this entity and ensuring that family members are screened.
  • radiologists may be the first to recognize the risk of surgery on an organ without assessing the status of other potential sites of disease, particularly pheochromocytoma and CNS hemangioblastomas (25).
  • VHL disease and associated disorders e.g. tumors and cysts
  • markers and therapeutic agents will be most useful in management and treatment of more common sporadic counterpart tumors.
  • the present invention relates to methods for the diagnosis of VHL and sporadic renal cell carcinoma and other VHL and sporadic counterpart tumors by detecting the co-expression of EPO and EPO receptors (EPOR) in tissues and fluids obtained from patients, particularly patients with VHL disease or patients at risk for developing VHL disease.
  • the present invention relates to the treatment of patients with such tumors.
  • the present invention includes the diagnosis and treatment of non-tumor lesions in patients co-expressing EPO and EPO receptors in tissues and fluids of such patients.
  • the invention relates to method for the diagnosis of VHL and sporadic renal cell carcinoma and other VHL and sporadic counterpart tumors in an mammal, comprising
  • the biological sample can be typically derived e.g. from renal, neural, pancreatic or neuroendocrine tissue obtained by biopsy.
  • the biological sample can be obtained from blood serum, cerebral spinal fluid, cyst fluid and urine.
  • the methods for determining the co-expression of EPO and EPOR include a detectably labeled ligand-receptor binding assays, detectably labeled immunocytochemical assays, detectably labeled flow cytometric techniques, protein chemistry and RT-PCR.
  • the invention relates to a method for the diagnosis of
  • VHL and sporadic renal cell carcinoma and other VHL and sporadic counterpart tumors in an mammal comprising
  • the invention relates to a method for the diagnosis of VHL and sporadic renal cell carcinoma and other VHL and sporadic counterpart tumors in an mammal, comprising (a) obtaining a biological sample from the mammal; and
  • the invention relates to the treatment of patient with VHL and sporadic renal cell carcinoma and other VHL and sporadic counterpart tumors comprising the administration to a patient in need of such treatment a pharmaceutically effective amount of a protein-based (e.g., antibody or EPO analogues) or small molecule antagonist of the EPOR (i.e., binds to the receptor but does not activate the receptor).
  • a protein-based e.g., antibody or EPO analogues
  • small molecule antagonist of the EPOR i.e., binds to the receptor but does not activate the receptor.
  • the invention relates to the treatment of patient with VHL and sporadic renal cell carcinoma and other VHL and sporadic counterpart tumors comprising the administration to a patient in need of such treatment a pharmaceutically effective amount of nucleic acid which acts to downregulate the expression of EPO and/or the EPOR.
  • Figure 1 represents photographs of histological sections wherein positive cytoplasmic immunohistochemistry staining for EPO is detected in cells of solid RCC (A), cystic RCC (B), microscopic RCC arising in a cyst, (C), and benign cyst (D).
  • A solid RCC
  • B cystic RCC
  • C microscopic RCC arising in a cyst
  • D benign cyst
  • Figure 2 represents photographs of histological sections wherein positive cytoplasmic immunohistochemistry staining for EpoR is detected in cells of solid RCC (A), cystic RCC (B), microscopic RCC arising in a cyst, (C) and benign complex cyst (D).
  • A solid RCC
  • B cystic RCC
  • C microscopic RCC arising in a cyst
  • D benign complex cyst
  • FIG. 3 represents photographs of various wherein (A) Representative four VHL-associated RCCs (Tl-4) show marked allelic imbalance (loss of heterozygosity) after amplification with VHL gene flanking primers D3S1110 and D3S2452. Matched adjacent normal kidney (N 1-4) shows preservation of both alleles of the VHL gene; (B) Expression of EPO and EPOR mRNA in VHL-associated RCC and normal kidney by RT-PCR; Lane 1 -molecular weight marker, lane 2-normal VHL kidney, lane 3 -VHL-associated RCC. EPO mRNA is expressed in RCC; Normal kidney shows low intensity of Epo mRNA expression.
  • EPOR mRNA is expressed in VHL- associated RCC but not in normal kidney;
  • C Western blot analysis for Epo and EpoR expression in VHL-associated RCC; EPO and EPOR proteins are expressed in three VHL-associated RCC (lanes 1-3) and in VHL-associated HB (lane 4) used as positive control; Adrenal gland tissue is used as negative control (lane 5).
  • Figure 4 represents photographs and diagrammatic representations of the histogenesis of VHL tumors, wherein similar histopathology of HB (A) and low grade RCC (B) in VHL disease (H&E): both tumors are composed of clear cells intermixed with numerous small vessels.
  • C Principal events of early nephrogenesis (modified after Horster et al., 15).
  • Ureteric bud which is derived from Wolffian duct, invades nephrogenous mesenchyme and initiates reciprocal signaling between epithelial (ductal) and mesenchymal cell types.
  • Nephrogenous mesenchyme expresses stem cells of several cell lineages including mesenchymal, epithelial and stromogenic ones.
  • Figure 5 represents the nucleotide sequence (SEQ ID NO: 1) encoding human erythropoietin (EPO).
  • Figure 6 represents the nucleotide sequence (SEQ ID NO: 2) encoding human erythropoietin receptor (EPOR).
  • Figure 7 represents the forward primer nucleotide sequence (SEQ ID NO: 3) ofEPO for PCR.
  • Figure 8 represents the reverse primer nucleotide sequence (SEQ ID NO: 4) of EPO for PCR.
  • Figure 9 represents the forward primer nucleotide sequence (SEQ ID NO: 5) of EPOR for PCR.
  • Figure 10 represents the reverse primer nucleotide sequence (SEQ ID NO: 6) ofEPOR for PCR.
  • Figure 11 represents the reverse primer nucleotide sequence (SEQ ID NO: 7) of glucose phosphate isomerase (GPI) for PCR.
  • VHL-associated tumors The histogenesis of VHL-associated tumors remains controversial. In HB, several different cell types have been implicated as the cell of origin (12-15) and in RCC an uncertainty persists between proximal versus distal tubular cell of origin (16- 18). Studies suggest that VHL-associated HB derives from primitive embryonal angiomesenchymal cells capable of differentiating into hemangioblasts (9-11). Primitive blood islands express EPOR (19) and co-expression of EPO because of VHL deficiency may facilitate uncontrolled cell growth.
  • VHL-associated HB and RCC are strikingly similar morphologically, and they are composed of clear cells intermixed with numerous small vessels (Fig 4 A, B).
  • Fig 4 A, B Like HB, renal lesions are multifocal and share the same genetic abnormality (8).
  • LOH of the wild-type VHL allele has been detected in multiple RCC as well as in many benign cysts in kidney parenchyma of VHL patients, suggesting that some renal cysts are precursors of RCC (8).
  • Activation of the HIF-I pathway as a mechanism of angiogenesis is present in HB and RCC.
  • HB and low grade RCC have almost identical morphology on H&E staining, the presence of epithelial markers in RCC suggested commitment to epithelial differentiation. Interestingly, some HBs also express epithelial markers (20).
  • VHL-associated HB derives from primitive embryonal angiomesenchymal cells capable of differentiating into hemangioblasts (9).
  • RCC may contain extramedullary hematopoiesis (21), a possible indicator of retained angiomesenchymal differentiation in RCC.
  • nephrogenous mesenchyme and angiomesenchyme are derived from the same mesodermal precursor (22, 23) (Fig. 4C).
  • nephrogenic mesenchyme has a pluripotential capacity and gives rise to renal epithelia, mesenchymal and stromal elements (23).
  • EPO in RCC and renal cysts results from VHL gene deficiency.
  • EPOR expression normally occurs in the angioblast stage of embryonic development as part of hypoxia response. During normal development such EPOR expression in cells is transient. However, because of inactivation of VHL protein in cells of a VHL patient, EPO and EPOR co-expression may persist in tumor precursor cells (9). The retention of EPO and EPOR co-expression in primitive mesenchymal cells may lead to cell proliferation via the autocrine stimulation (24) and become a critical pathogenetic step in tumor formation. Therefore, co-expression of EPO and EPOR in VHL-associated RCC and cysts suggests the possibility that a precursor cell of renal lesions is a developmentally arrested, pluripotential embryonal cell derived from nephrogenous mesenchyme.
  • VHL gene deficiency causes precursor cells in nephrogenous mesenchyme to arrest early in the developmental stage and to co- express EPO and EPOR; EPO and EPOR co-expression persists and is detectable in benign renal cysts and RCC.
  • Analogies between HB and RCC, including EPO and EPOR co-expression, morphology, and mesenchymal derivation indicate a similar developmental origin for both VHL tumor types.
  • the present teachings provide the first evidence that EPO and/or the EPO or EPOR plays a role in human VHL disease development, and more importantly, the ligand and receptor can potentially be used as diagnostic markers or as therapeutic targets for VHL disease associated cancer and/or cyst treatment.
  • biological sample means a sample of cells from a patient. These cells may be part of a tissue or organ sample obtained, for example, by biopsy, or they may be specimens of fluid. Preferably, such samples are obtained from easily obtained sources such as peripheral blood or urine. Such samples are commonly obtained by drawing a blood sample from a patient and then using standard techniques to purify or partially purify the fluid from the blood. For example, the plasma portion of the blood forms a layer above the buffy coat. Fractions from blood can also be isolated in a variety of other ways. Other specimens of fluid are cerebral spinal fluid and cyst fluid. Tissues for biopsy can be obtained from renal, neural, pancreatic or neuroendcrine.
  • the biological samples may be of normal cells, or may be of tumor cells, the tumor cells being benign or malignant.
  • an assay that uses cells from such biological samples, herein will be used to determine the presence of EPO and EPOR transcripts or proteins, or levels of EPO and/or EPOR transcripts or proteins.
  • the "test sample” will generally be a sample for which the presence or level of EPO or EPOR are unknown and are being tested to provide, for example, an indication of the presence of the tumor cells.
  • a "control sample” will preferably also be used.
  • the control sample can be from normal (i.e., non-tumorigenic or non-neoplastic) tissue from the same patient from which the test sample is taken or can be from another person known or thought not to have the tumor that is present or thought to be present in the patient from whom the test sample is taken.
  • the control sample comprises the same type of cells that comprise the test sample.
  • the test sample comprises renal cells, it is preferable that the control sample is also a renal sample.
  • Sporadic counterpart tumors can include, but are not limited to, hemangioblastoma of the central nervous system and retina, pheochromocytoma, pancreatic microcystic adenoma, pancreatic neuroendocrine tumors, epidiymal cystadenoma and endolymphatic sac tumors. Samples may be obtained from all such tumor tissues.
  • the biological samples can be obtained from patients at various times.
  • a sample may be obtained from an individual who is suspected of having a tumor or cancer.
  • Assay of such sample using the methods described below can indicate whether the individual has a tumor or cancer.
  • Samples may also be obtained from an individual known to have a tumor or cancer (i.e., the sample is taken after the patient has already been diagnosed). Assay of such sample using the described methods may have prognostic value to the individual.
  • Multiple samples can also be taken from the same individual, for example, at different times after diagnosis. Assay of such samples can indicate whether the cancer or tumor is growing or spreading.
  • Such assays on multiple samples are especially informative in the case where it is desired to determine the effect of a chemotherapeutic, other therapeutic agents or surgical resection on the growth and progression of the tumor or cancer in the individual.
  • test samples are preferably obtained from patients who are known to have or suspected of having a tumor or cancer. Methods for diagnosis of particular tumors or cancers in patients are well known in the art of medicine, oncology and hematology.
  • saying when used in reference to biological samples, preferably the cells in biological samples, refers to assessment or measurement of the presence and/or levels or concentrations of EPO and EPOR gene expression (transcripts or protein isoforms) in the samples. This assessment is done by detecting and/or measuring the levels of RNA transcribed from the EPO and EPOR genes or proteins which are translated from the RNA transcripts.
  • multiple transcripts called alternative transcripts in the art, may be present when either EPO or the EPOR gene is transcribed.
  • Such alternative transcripts come from different combinations of exons encoded by the EPO or EPOR genes. Some or all of these transcripts are translated to produced EPO orEPOR proteins.
  • the EPO and EPOR proteins that are obtained from translation of different alternative transcripts may be somewhat different (in size and/or sequence) from one another depending on the combination of exons existing in the particular alternative transcript or on how the exon sequences in the transcripts are translated. Such different proteins are known in the art as protein "isoforms.”
  • Assaying these samples may involve detection or quantification of one or more specific alternative transcripts or protein isoforms. For example, one may be interested in determining the presence, level or concentration of one specific alternative transcript or protein isoform.
  • assaying the samples may involve detection or levels of EPO or EPOR transcripts or proteins as a whole. For example, in determining the level or concentration of EPO or EPOR transcripts, the sum of the levels of all of the different alternative transcripts or protein isoforms may be used.
  • “elevated” means an increase in the amount of the transcript or isoform in the test sample as compared to the control sample.
  • “Elevated in the test sample as compared to the control sample” describes a situation where the presence of EPO or EPOR transcripts or proteins is detected in the test sample and the amount, level or concentration of the EPO or EPOR transcripts or proteins in the test sample is greater than in the control sample. This means, in the control sample, that EPO OR EPOR transcripts or proteins are detected, but are not present in amounts, levels or concentrations as high as are present in the test sample.
  • a comparison of the levels in the test sample to the levels in one or more control samples is performed.
  • Levels in a control sample or samples can be represented by a single value or range of values.
  • an average of the EPO or EPOR levels in more than one control sample is used for comparison with the EPO or EPOR levels in the test sample. More preferably, an average of the EPO or EPOR levels from a number of control samples sufficient to provide a statistically significant comparison with EPO or EPOR levels present in the test sample is used.
  • the control sample levels of EPO or EPOR may be determined at the same time at which EPO or EPOR levels in the test sample is determined.
  • the EPO or EPOR levels in the control samples may also be predetermined, meaning that the levels have been determined before the time at which EPO or EPOR levels in the test samples are determined.
  • the values are preferably normalized or standardized such that they can be legitimately compared with values for EPO or EPOR levels in test samples that are determined later.
  • the amount of the increase can be of various magnitudes.
  • the increase may be relatively large. For example, a large increase could be a 100% or more increase in EPO or EPOR expression in the test sample as compared to the control sample. However, the increase may be relatively small. For example, the increase may be less than 100%, less than 50%, or even less than a 10% increase of the transcript or protein in the test sample as compared to the control sample. Preferably, whatever the degree or magnitude of the increase, such increase is statistically significant. Methods for determining whether an increase is statistically significant are well known in the art of statistics and probability.
  • Comparison of the test sample to the control sample for the presence and/or levels of EPO or EPOR expression is used to characterize the tumor or cancer, i.e., to determine the "aggressiveness" of the tumor or cancer.
  • a level of EPO or EPOR transcripts or proteins in the test sample that is higher than the level in the control sample indicates presence of an aggressive tumor or cancer.
  • the extent or degree of the increase between the level of EPO or EPOR transcripts or proteins in the test sample and the control sample correlates with degree of aggressiveness of the tumor or cancer. Aggressiveness refers to the nature of tumor cell growth in a patient. For example, an aggressive cancer has a higher probability of producing an unfavorable outcome in a patient than a cancer that is less aggressive.
  • Unfavorable outcome normally refers to the probability that a patient will have a relatively short lifespan due to the aggressive nature of the cancer. Patients with a less aggressive cancer or cancer that is not aggressive are expected to have a longer lifespan than a patient with an aggressive form of the cancer. EPO and EPOR Co-Expression Associated with VHL and Sporadic Renal Cell Carcinoma, Other Sporadic Counterpart Tumors and Renal Cysts:
  • Methods are provided to determine the level of EPO or EPOR gene expression in a biological sample.
  • One method determines the amount of RNA transcribed from the EPO or EPOR gene.
  • Other method determines the amount of EPO or EPOR protein, including detectably labeled ligand-receptor binding assays, detectably labeled immunocyochemistry, protein chemistry and detectably labeled flow cytometric techniques.
  • RNA is first isolated from the tissue or cells comprising the biological sample.
  • the biological sample is preferably a biopsy sample of tissue or a fluid sample containing EPO or EPOR as was described earlier.
  • a variety of methods of RNA isolation from cells and/or tissues is well known to those skilled in the art. Any of such methods can be used.
  • One such method uses the Trizol RTM reagent from Gibco BRL. Such methods isolate total cellular RNA. Other methods isolate polyadenylated RNA. Methods that provide either type of RNA can be used.
  • Reverse transcriptase reactions coupled to polymerase chain reactions is one method to assay for the presence of an RNA in a pool of total RNA from a tissue or cell. Detection of a particular RNA is dependent on primers used in the PCR reaction.
  • the initial step in RT-PCR is a reverse transcription step.
  • Procedures for reverse transcription are well known to those skilled in the art and a variety of procedures can be used.
  • Either total RNA or polyadenylated niRNA can be used as the template for synthesis of cDNA by the reverse transcriptase enzyme.
  • oligo(dT) is used as the primer in the reverse transcription reaction. Oligo(dT) hybridizes to the ⁇ oly(A) tails of mRNAs during first strand cDNA synthesis. Since all mRNAs normally have a poly(A) tail, first strand cDNA is made from all mRNAs present in the reaction (i.e., there is no specificity).
  • specific primers are used in place of oligo(dT) and specific KNAs are reverse transcribed into DNA. The specific primers preferably are complementary to a region near the 3' end of the RNA in order that full length or nearly full length cDNA is produced. Primer selection is preferably made using the guidelines described below for selection of PCR primers. A number of different primers can be used with good results. Another embodiment can be given by the use of random primers.
  • the reverse transcriptase enzyme used in the reaction is stable at temperatures above 60 degree C, for example, Superscript II RT (Gibco BRL).
  • MMLV reverse transcriptase can also be used.
  • the reverse transcriptase reaction mixture contains 10 mM Tris (pH 8.3), 40 mM KCl, 1.5 mM MgCl 2 , 1 mM dithiothreitol, 200 uM each of dATP, dCTP, dTTP and dGTP, 200 ng of the primer, 10 U .of AMV reverse transcriptase from Boehringer Mannheim Biochemicals, and 20 units of RNASIN from Promega.
  • Trehalose is a disaccharide that has been shown to stabilize several enzymes including RT at temperatures as high as 60 degree C (Mizuno, et al., Nucleic Acids Res.
  • Trehalose addition allows the use of high temperatures in the reverse transcription reaction (e.g., as high as 60 degree C). Therefore, trehalose can be added to the reverse transcriptase reaction such that it is present in a final concentration of between 20 to 30%.
  • the reverse transcriptase reaction is then performed at a temperature between 35 to 75 degree C, more preferably at a temperature from between 50 to 75 degree, most preferably at a temperature of 60 degree C.
  • PCR Once the reverse transcriptase reaction is carried out, the cDNA produced is amplified by PCR. In one embodiment, the entire RT-PCR reaction is carried out on a standard thermal cycler according to the methods described in the GeneAmp RNA PCR kit obtained from Perkin-Elmer/Cetus, for example. A 0.5 pg sample of total RNA from the cells is used to produce the first strand cDNA.
  • the amplification cycle protocol is as follows: 95 degree C for 2 minutes, 95 degree C for 1 minute, 56 degree C for 1 minute, and 72 degree C for 2 minutes, through 35 cycles. The annealing temperature depends on the primers used.
  • a standard PCR reaction contains a buffer containing
  • high stringency PCR conditions refers to conditions that do not allow base- pairing mismatches to occur during hybridization of primer to template. Such conditions are equivalent to or comparable to denaturation for 1 minute at 95 degree C in a solution comprising 10 mM Tris-HCl (pH 8.3), 50 mM KCl, and 6.0 mM MgCl 2 , followed by annealing in the same solution at about 62 degree C for 5 seconds.
  • the products of the PCR reaction can be detected in various ways.
  • One way is by agarose gel electrophoresis which involves separating the DNA in the PCR reaction by size in electrophoresis. The agarose gel is then stained with dyes that bind to DNA and fluoresce when illuminated by light of various wavelengths.
  • the dye used is ethidium bromide and the illumination uses an ultraviolet light
  • One primer is located at each end of the region to be amplified.
  • Such primers will normally be between 10 to 30 nucleotides in length and have a preferred length from between 18 to 22 nucleotides.
  • the smallest sequence that can be amplified is approximately 50 nucleotides in length (e.g., a forward and reverse primer, both of 20 nucleotides in length, whose location in the sequences is separated by at least 10 nucleotides). Much longer sequences can be amplified.
  • the length of sequence amplified is between 75 and 250 nucleotides in length.
  • One primer is called the "forward primer" and is located at the left end of the region to be amplified.
  • the forward primer is identical in sequence to a region in the top strand of the DNA (when a double-stranded DNA is pictured using the convention where the top strand is shown with polarity in the 5' to 3' direction).
  • the sequence of the forward primer is such that it hybridizes to the strand of the DNA which is complementary to the top strand of DNA.
  • the other primer is called the "reverse primer” and is located at the right end of the region to be amplified.
  • the sequence of the reverse primer is such that it is complementary in sequence to a region in the top strand of the DNA.
  • the reverse primer hybridizes to the top strand of the DNA
  • PCR primers should also be chosen subject to a number of other conditions. PCR primers should be long enough (preferably 10 to 30 nucleotides in length) to minimize hybridization to greater than one region in the template. Primers with long runs of a single base should be avoided, if possible. Primers should preferably have a percent G+C content of between 40 and 60%. If possible, the percent G+C content of the 3' end of the primer should be higher than the percent G+C content of the 5' end of the primer. Primers should not contain sequences that can hybridize to another sequence within the primer (i.e., palindromes). Two primers used in the same PCR reaction should not be able to hybridize to one another. Although PCR primers are preferably chosen subject to the recommendations above, it is not necessary that the primers conform to these conditions. Other primers may work, but have a lower chance of yielding good results.
  • PCR primers that can be used to amplify DNA within a given sequence are preferably chosen using one of a number of computer programs that are available. Such programs choose primers that are optimum for amplification of a given sequence (i.e., such programs choose primers subject to the conditions stated above, plus other conditions that may maximize the functionality of PCR primers).
  • One computer program is the Genetics Computer Group (GCG recently became Accelrys) analysis package which has a routine for selection of PCR primers.
  • GCG Genetics Computer Group
  • One such web site is http://alces.med.umn.edu/rawprimer.html.
  • Another such web site is http://www-genome.wi.mit.edu/cgi-bin/primer/- ⁇ rimer3_www.cgi.
  • Forward and reverse primers can be selected from a variety of regions of the EPO and EPOR genes. Actually, a very large number of primers can be designed using the sequence of the EPO or EPOR genes and such probes successfully used.
  • the forward primer is designed using coding sequence for EPO and for EPOR and the reverse primer is designed using a near coding sequence of EPO and EPOR.
  • the PCR procedure can also be done in such a way that the amount of PCR products can be quantified.
  • Such "quantitative PCR” procedures normally involve comparisons of the amount of PCR product produced in different PCR reactions.
  • a number of such quantitative PCR procedures, and variations thereof, are well known to those skilled in the art.
  • One inherent property of such procedures ⁇ however, is the ability to determine relative amounts of a sequence of interest within the template that is amplified in the PCR reaction.
  • Real-time PCR utilizes a thermal cycler (i.e., an instrument that provides the temperature changes necessary for the PCR reaction to occur) that incorporates a fluorimeter (i.e. an instrument that measures fluorescence).
  • a thermal cycler i.e., an instrument that provides the temperature changes necessary for the PCR reaction to occur
  • a fluorimeter i.e. an instrument that measures fluorescence
  • the reaction mixture also contains a reagent whose incorporation into a PCR product can be quantified and whose quantification is indicative of copy number of that sequence in the template.
  • SYBR Green I Molecular Probes, Inc.; Eugene, Oreg.
  • SYBR Green I Molecular Probes, Inc.; Eugene, Oreg.
  • SYBR Green I Molecular Probes, Inc.; Eugene, Oreg.
  • resulting DNA products bind SYBR Green I and fluoresce.
  • the fluorescence is detected and quantified by the fluorimeter.
  • Such technique is particularly useful for quantification of the amount of template in a PCR reaction.
  • two different primers are preferably alternatively used.
  • the first primer is called a EPO forward primer and has the sequence 5 1 - TCTATGCCTGGAAGAGGATGGAGGTCG -3' (SEQ ID NO. 3).
  • the second primer is called EPO Reverse primer and has the sequence 5'- TGCGGAAAGTGTCAGCAGTGATTGTTC -3' (SEQ ID NO. 4).
  • GPI Exon3R the primer preferably is called GPI Exon3R and has the sequence 5'- TCGGTGTAGTTGATCTTCTC-3' (SEQ ID NO. 7).
  • the first primer is called a EPOR forward primer and has the sequence 5'- CACAAGGGTAACTTCCAGCTGTGGCTGTA -3' (SEQ ID NO. 5).
  • the second primer is called EPOR Reverse primer and has the sequence 5'- CATTTGTCCAGCACCAGATAGGTATCCTGG -3' (SEQJD NO. 6).
  • glucose phosphate isomerase the primer preferably is called GPI Exon3R and has the sequence 5'- TCGGTGTAGTTGATCTTCTC-3' (SEQ ID NO. 7).
  • a preferred variation of real-time PCR is TaqMan MGB (Applied Biosystems) PCR.
  • the basis for this method is to continuously measure PCR product accumulation using a dual-labeled flourogenic oligonucleotide probe called a TaqMan MGB probe.
  • the "probe” is added to and used in the PCR reaction in addition to the two primers.
  • This probe is composed of a short (ca. 20-30 bases) oligodeoxynucleotide sequence that hybridizes to one of the strands that are made during the PCR reaction. That is, the oligonucleotide probe sequence is homologous to an internal target sequence present in the PCR amplicon.
  • the probe is labeled or tagged with two different flourescent dyes.
  • reporter dye On the 5 1 terminus is a "reporter dye” and on the 3' terminus is a “quenching dye.”
  • One reporter dye that is used is called 6-carboxy fluorescein (FAM).
  • One quenching dye that is used is called 6-carboxy tetramethyl-rhodamine (TAMRA).
  • FAM 6-carboxy fluorescein
  • TAMRA 6-carboxy tetramethyl-rhodamine
  • the probe is cleaved by the 5' nuclease activity of Taq polymerase, thereby releasing the reporter from the oligonucleotide-quencher and producing an increase in reporter emission intensity. During the entire amplification process the light emission increases exponentially.
  • the instrument used to detect the fluorescence is preferably an ABI Prism 7700, which uses fiber optic systems that connect to each well in a 96-well PCR tray format.
  • the laser light source excites each well and a CCD camera measures the fluorescence spectrum and intensity from each well to generate real-time data during PCR amplification.
  • the ABI 7700 Prism software examines the fluorescence intensity of reporter and quencher dyes and calculates the increase in normalized reporter emission intensity over the course of the amplification. The results are then plotted versus time, represented by cycle number, to produce a continuous measure of PCR amplification. To provide precise quantification of initial target in each PCR reaction, the amplification plot is examined at a point during the early log phase of product accumulation.
  • RNA that is transcribed from the EPO or EPOR gene can be detected using other procedures.
  • One such method is known as Northern blot hybridization.
  • RNA is isolated from colorectal tissues or leukocytes and separated by size using gel electrophoresis. The RNAs in the gel are then transferred to a membrane. After transfer of the RNA to the membrane, a nucleotide probe is labeled and hybridized to the RNA on the membrane. Hybridization of a DNA probe to RNA on the membrane is detected by autoradiography or chemiluminescence.
  • RNA is applied directly to a membrane.
  • the nucleotide probe is then labeled and hybridized to the RNA on the membrane. Hybridization is detected by autoradiography or chemiluminescence. Probes of many different lengths and sequences can be designed and used in Northern blotting experiments to detect EPO or EPOR transcripts.
  • Protein or cell-free extracts are made from tumor tissues or fluids.
  • cells are lysed in 500 ul ice-cold Lysis Buffer (50 mM Tris pH 7.5; 1% Triton X-IOO; 100 mM NaCl; 50 mM NaF; 200 uM Na 3 VO 4 ; 10 ug/ml pepstatin and leupeptin) (all chemicals from Sigma Chemical Co., St. Louis, Mo.) for approximately 30 min at 4 degree C.
  • the cell lysate suspension is then microcentrifuged at 4 degree C. (14,000 RPM for 10 min). The supernatant is removed and stored at —80 degree C.
  • Proteins are separated using SDS- polyacrylamide gel electrophoresis (SDS-PAGE) through a 6%-7.5% acrylamide gel at 100V.
  • the samples are transferred to an Immobilon-P membrane (Millipore, Bedford, Mass.). Blots are blocked in phosphate buffered saline (PBS: 138 mM NaCl, 15 mM Na 2 HPO 4 , 1.5 mM KCl, and 2.5 mM KH 2 PO 4 ), containing 5% non-fat dehydrated milk and 0.1% Tween-20 (Sigma Chemical Co., St. Louis, Mo.) overnight at 4 degree C.
  • PBS phosphate buffered saline
  • Tween-20 Sigma Chemical Co., St. Louis, Mo.
  • Blots are incubated for 90-120 min at room temperature in PBS with primary anti-EPO or EPOR antibody and then washed three times in PBS with 0.1% Tween-20. Blots are then incubated with secondary antibody conjugated to horseradish peroxidase (1:4000 dilution) (Sigma Chemical Co., St. Louis, Mo.) for 1 hour at room temperature and washed again as described above. Signal is visualized by incubating with Super Signal chemiluminescent substrate (Pierce, Rockford, 111.) and exposing the membrane to Kodak scientific imaging film (Kodak, Rochester, N.Y.).
  • EPO or EPOR protein refers not to a single protein, but to multiple EPO or EPOR proteins could be represented by different isoforms.
  • the present invention utilizes antibodies that are immunospecific for the EPO or EPOR protein.
  • immunospecific means the antibodies have greater affinity for the EPO or EPOR protein than for other proteins.
  • affinity of the antibodies for EPO or EPOR protein is many fold greater than their affinity for any other proteins.
  • the EPO or EPOR antibodies do not have affinity for any proteins other than EPO or EPOR protein.
  • antibody encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity or specificity.
  • Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments.
  • Antibodies raised against EPO or EPOR are produced by immunizing a host animal with a EPO or EPOR protein or an antigenic fragment thereof.
  • Suitable host animals for injection of the protein immunogen include, but are not limited to, rabbits, mice, rats, goats, and guinea pigs.
  • Various adjuvants may be used to increase the immunological response of the immunogen or antigen (i.e., the EPO or EPOR protein or peptide) in the host animal.
  • the adjuvant used depends, at least in part, on the host species. For example, guinea pig albumin is commonly used as a carrier for immunizations in guinea pigs.
  • Such animals produce heterogeneous populations of antibody molecules, which are referred to as polyclonal antibodies and which may be derived from the sera of the immunized animals. Such sera may be used directly, or the specific antibodies desired can be purified from the sera, using methods well known to those of skill in the art.
  • Antibodies are also prepared using an oligopeptide having a sequence which is identical to a portion of the amino acid sequence of a EPO or EPOR protein isoform.
  • the oligopeptide has an amino acid sequence of at least five amino acids, and more preferably, at least 10 amino acids that are identical to a portion of the amino acid sequence of a EPO or EPOR protein.
  • Such peptides are conventionally fused with those of another protein such as keyhole limpet hemocyanin and antibody is produced against the chimeric molecule.
  • Such peptides can be determined using software programs, for example the Mac Vector program, to determine hydrophilicity and hydrophobicity and ascertain regions of the protein that are likely to be present at the surface of the molecule.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally- occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site, also called epitope. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method, first described by Kohler and Milstein (Nature 256:495-497, 1975), in which case the hybridoma cell lines that are obtained secrete the monoclonal antibodies during growth.
  • hybridomas that secrete monoclonal antibodies are made by injecting mice with the desired antigen.
  • the antigens frequently are peptide antigens which are chosen using similar procedures as described above for selection of peptide antigens for making polyclonal antibodies.
  • spleen cells are taken from the immunized mice and are fused to myeloma cells. Clones of fusion cells are then obtained and are screened for production of anti-EPO or EPOR antibodies.
  • immunoassays may be used for screening to identify antibodies having the desired specificity. These include protocols which involve competitive binding or immunoradiometric assays and typically involve the measurement of complex formation between the respective EPO or EPOR protein and the antibody.
  • the hybridoma cell lines may be grown in cell culture and culture medium containing the monoclonal antibodies collected.
  • the hybridoma cell lines may be injected into, and grown within, the peritoneal cavity of live animals, preferably mice.
  • the monoclonal antibodies are secreted.
  • This peritoneal fluid, called "ascites,” is collected using a syringe to obtain the monoclonal antibodies.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, Iga, IgD and any class thereof.
  • Antibody preparations may be isolated or purified.
  • An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody may be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step
  • Antibodies immunospecific for EPO or EPOR are useful for identifying colorectal tissues or leukocyte cells that express EPO or EPOR proteins.
  • the diagnostic/prognostic methods comprise the steps of contacting tissues or fluids with such antibody and assaying for the formation of a complex between the antibodies and a EPO or EPOR protein in the samples.
  • the cells are permeabilized.
  • Interactions between antibodies and a protein or peptide in the sample are detected by radiometric, colorimetric, or fluorometric means.
  • Detection of the antigen-antibody complex may be accomplished by addition of a secondary antibody that is coupled to a detectable tag, such as for example, an enzyme, fluorophore, or chromophore.
  • the detection method employs an enzyme-linked immunosorbent assay (ELISA), Western immunoblot procedure and/or immunoprecipitation and/or flow cytometric techniques
  • detectably labeled has the ordinary meaning in the art.
  • a molecule e.g., antibody or polynucleotide probe
  • an atom e.g., radionuclide
  • molecule e.g., fluorescein
  • complex e.g., fluorescein
  • reporter molecules e.g., a biomolecule such as an enzyme
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Labels useful in the present invention include biotin for staining with labeled avidin or streptavidin conjugate, magnetic beads (e.g., Dynabeads TM), fluorescent dyes (e.g., fluorescein, fluorescein- isothiocyanate [FITC], Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX [Amersham], SyBR Green I & II [Molecular Probes], and the like), radiolabels'(e.g., .sup.3 H, .sup.125 I, .sup.35 S, .sup.14 C, or .sup.32 P), enzymes (e.g., hydrolases, particularly phosphata
  • Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • Means of detecting such labels are well known to those of skill in the art.
  • radiolabels and chemiluminescent labels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted light (e.g., as in fluorescence- activated cell sorting).
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected by observing the color associated with the label.
  • Immunocytochemistry Suitable methods of cell or tissue preparation and for binding antibodies to
  • EPO or EPOR include those used for conventional immunocytochemistry, and are well known in the art.
  • Immunophenotyping e.g., ICC
  • Staines 1988, J. Histochem. Cytochem. 36:145
  • Gillitzer et al. 1990, J. Histochem. Cytochem. 38:307
  • Wagner and Worman 1988, Stain Technology 63:129
  • McGovern and Crocker 1987, Am. J. Clin. Pathol. 88, 480;
  • the cells of interest are fixed (e.g., in buffered formalin) or permeablized (e.g., using by agents such as buffered detergent solution, e.g., Tween 20, Triton X, or NP 40 [ ⁇ 0.5% v/v] in Tris- or phosphate based buffers).
  • Fixation or permeabilization is particularly preferred when the target antigen is not an extracellular protein (e.g., when the target is a cytoplasmic protein) to increase the accessibility of the antigen to the antibody.
  • any unbound antibody is removed in a wash step, e.g., PBS (phosphate buffered saline) or Tris-based buffer with or without non-ionic detergent.
  • radioligand binding assays can be performed in tubes containing binding buffers (such as, 50 mM TRISHCL, pH 7.4, 10 mM MgCl.sub.2, 0.25% BSA), protease inhibitors (5.mu.g/ml leupeptin, 5 .mu.g/ml aprotinin, 100 .mu.g/ml bacitracin, and 100 .mu.g/ml benzamidine), cold competitor (EPOR binding agent, uM final concentration), [ 3 H] EPOR binding agent (1-50 nM).
  • binding buffers such as, 50 mM TRISHCL, pH 7.4, 10 mM MgCl.sub.2, 0.25% BSA
  • protease inhibitors 5.mu.g/ml leupeptin, 5 .mu.g/ml aprotinin, 100 .mu.g/ml bacitracin, and 100 .mu.g/ml benzamidine
  • Binding reactions are initiated by adding 60 ug amount of membrane protein (ranging from e.g 60 to 100 ug) in a volume which can range from e.g. 250ul to ImI. All incubations can be carried out at 4°C if an antagonist radioligand is used or at room temperature if an agonist radioligand is used for an incubation time which allows the reaching of the equilibrium. Free radioligand is separated from bound ligand by rapid filtration through a glass fiber filter using a cell harvester. The filter disks are then washed several times with cold (4 degree C) binding buffer lacking BSA prior to counting. Samples from tissue biopsies or fluids are compared to normal controls.
  • the present invention also relates to the treatment of a patient with renal cell carcinoma, other VHL sporadic counterpart tumors and renal cysts comprising the administration to a patient in need of such treatment a pharmaceutically effective amount of any agent which interferes with the binding of EPO to its receptor and the activation of that receptor.
  • the present invention also relates to the treatment of a patient with VHL disease comprising the administration to a patient in need of such treatment a pharmaceutically effective amount of a protein-based (e.g., antibody or EPO analogues) or small molecule antagonist of the EPOR (i.e., binds to the receptor but does not activate the receptor).
  • EPO analogues can be found in US Patent Nos: 5,106,954; 5,292,654; 5,548,064; 5,712,370; 6,153,407; and 6,187,564, which are hereby incorporated by reference in their entirety.
  • the invention relates to the treatment of patient with VHL disease comprising the administration to a patient in need of such treatment a pharmaceutically effective amount of nucleic acid which acts to downregulate the expression of EPO and/or the EPOR.
  • Nucleic acids which have the ability to downregulate the expression of EPO and EPOR include anti-sense nucleotides to the nucleic acid sequences which encode these proteins, most preferable interfering RNA's. Examples of the preparation of such interfering RNA's can be found in US Patent Publication Nos: 20030108923; 20020086356; 20030190654; 20040001811; and 20040038921, hereby incorporated by reference in their entirety.
  • Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections of RCC, renal cysts and adjacent normal kidneys. Tissue sections were first soaked in xylene and then washed in decreasing concentrations of ethanol. For antigen retrieval, sections were treated with DAKO Target Retrieval Solution and incubated at 95° C for 20-30 minutes. Sections were cooled at RT, washed three times in PBS, and then quenched for 20 minutes in a solution of 3 ml H 2 O 2 and 180 ml methanol. After three washes in PBS, sections were drenched in 10% horse serum for one hour.
  • the primary antibody was diluted in 2% horse serum, and the sections were incubated in a humidified chamber at 4° C overnight.
  • Primary antibodies used were rabbit polyclonal anti-human Epo, 1:100 and sheep polyclonal anti-human EpoR, 1 :200. Sections were then incubated with secondary antibody and avidin- biotin-complex for one hour each.
  • Diaminobenzidine (DAB) was used for visualization, followed by counterstaining with hematoxylin.
  • the sections were dehydrated with graded ethanol washes and xylene wash and mounted. The presence and intensity of antibody expression were examined in RCC, renal cysts and normal kidney.
  • Formalin-fixed, paraffin-embedded tissue from 11 RCC and normal kidney in 8 patients was used for LOH analysis. Unstained 5-niicron tumor tissue sections on glass slides were deparaffmized with xylene, rinsed in 100% to 80% ethanol, briefly stained with hematoxylin and eosin, and rinsed in 10% glycerol in TE buffer.
  • Microdissection was performed under direct light microscopic visualization using a 30-gauge needle. Control samples were obtained from the matched normal kidney tissue on the same histological slide. Procured cells were immediately resuspended in 30 ⁇ l buffer containing Tris-HCl, pH 8.0, 10 rnM ethylenediamine tetraacetic acid, pH 8.0, 1% Tween 20, and 0.1 mg/ml proteinase K, and were incubated at 37° C overnight. The mixture was boiled for 15 min to inactivate proteinase K and 2 ⁇ l of the DNA solution were used for PCR amplification using markers D3S2452 and D3S1110 (Research Genetics, Huntsville, AL) flanking the VHL gene.
  • P 32 -labeled amplified DNA was mixed with an equal volume of formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenole blue, and 0.05% xylene cyanol). Samples were then denatured for 5 minutes at 95°, loaded onto a 6% acrylamide gel, electrophoresed at 1800 V for 90 minutes, and transferred to Whatman paper for autoradiography.
  • formamide loading dye 95% formamide, 20 mM EDTA, 0.05% bromophenole blue, and 0.05% xylene cyanol.
  • RNAse-free DNAse I (0.2 U, total volume 10 ⁇ l) (Invitrogen) at RT for 15 min, denatured at 65 °C for 10 min, and subsequently reverse transcribed by Superscript II (Invitrogen) with 0.5 ⁇ g of oligo(dT) primer in a volume of 20 ⁇ l.
  • the PCR amplification was performed following cycling parameters: initial denaturing at 95°C for 1 min, 15 sec at 95 0 C, 30 sec at 68°C for 35 cycles.
  • Reaction mixture contained 4 ⁇ l of the cDNA template, 1.0 unit of Advantage-2 DNA polymerase (Clontech), IxPCR buffer, 200 ⁇ M of each dNTPs, and 200 nM of each primer.
  • the sequences of oligonucleotides used for RT-PCR were: forEpo, 5'- TCTATGCCTGGAAGAGGATGGAGGTCG -3' and 5 1 - TGCGGAAAGTGTCAGCAGTGATTGTTC -3'; for EpoR, 5'- CACAAGGGTAACTTCCAGCTGTGGCTGTA -3' and 5'- CATTTGTCCAGCACCAGATAGGTATCCTGG -3';
  • the PCR products were separated in 2.0% agarose gel, stained with ethidium bromide.
  • Frozen RCC tissue from three VHL patients was used for Western blot analysis for Epo and EpoR.
  • One frozen HB from a VHL patient was used as a positive control for Western blot analysis for both Epo and EpoR.
  • For Western blotting 20 ⁇ l of cell lysate were separated by electrophoresis on 4-12% gradient Bis- Tris gels (Invitrogen, Carlsbad, CA). Proteins were electro-transferred onto PVDF membrane (Novex, San Diego, CA).
  • EXAMPLE 1 Co-Expression of EPO and the EPO or EPOR in Cells of Renal Cell
  • RT-PCR consistently revealed the presence of Epo mRNA in VHL-associated RCC (Fig. 3B). In normal VHL kidney, Epo mRNA was expressed in low levels. EpoR mRNA was consistently detected by RT-PCR analysis in RCC, but not in normal kidney of VHL patients (Fig. 3B).
  • VHL renal tumors Co-expression of Epo and EpoR in VHL renal tumors was further confirmed by demonstrating both Epo and EpoR protein expression in VHL-associated RCC using Western blot analysis (Fig. 3C). VHL-associated HB was used as positive control and showed co-expression of both Epo and EpoR.
  • EXAMPLE 2 EPO is a important cytokine for maintenance of VHL tumor cells in culture and their differentiation into erythroid lineage cells.
  • VHL tumor cell line in culture condition was tested in the presence EPO.
  • EPO erythropoietin
  • EPOR erythropoietin receptor
  • EPO rich culture media culture the primary tumors was routinely accomplished and maintained in culture for other tests. These tumor cells were characterized and expanded to nucleated erythrocytes and mature erythrocytic progeny by varying the cell culture condition. These findings further support that EPO and EPOR functional pathway in these tumors is critical in the VHL tumor growth and differentiation.

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Abstract

L'invention concerne le diagnostic de la maladie de von Hippel-Lindau (VHL) et de carcinomes à cellules rénales sporadiques, ainsi que d'autres tumeurs homologues sporadiques associées à la maladie de VHL par détection d'EPO et de récepteurs d'EPO dans lesdites tumeurs et des tissus kystiques prélevés sur des patients, en particulier sur des patients souffrant de la maladie de VHL ou présentant un risque de développer cette maladie. L'invention concerne également le traitement de patients souffrant de ces tumeurs, kystes et lésions non tumorales.
PCT/US2005/033850 2004-09-20 2005-09-20 Methodes pour le diagnostic et le traitement de tumeurs et de carcinomes a cellules renales sporadiques associes a la maladie de von hippel-lindau (vhl), d'autres tumeurs et lesions non tumorales homologues sporadiques de la maladie de vhl qui co-expriment l'epo et le recepteur de l'epo Ceased WO2006034354A2 (fr)

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