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WO1996002002A1 - Analyse servant a deceler une augmentation du pouvoir envahissant de cellules epitheliales - Google Patents

Analyse servant a deceler une augmentation du pouvoir envahissant de cellules epitheliales Download PDF

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Publication number
WO1996002002A1
WO1996002002A1 PCT/IB1995/000577 IB9500577W WO9602002A1 WO 1996002002 A1 WO1996002002 A1 WO 1996002002A1 IB 9500577 W IB9500577 W IB 9500577W WO 9602002 A1 WO9602002 A1 WO 9602002A1
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cadherin
expression
prognostic
marker
catenin
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PCT/IB1995/000577
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English (en)
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Jack A. Schalken
Frans M. J. Debruyne
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Schalken Jack A
Debruyne Frans M J
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Application filed by Schalken Jack A, Debruyne Frans M J filed Critical Schalken Jack A
Priority to AU28971/95A priority Critical patent/AU2897195A/en
Publication of WO1996002002A1 publication Critical patent/WO1996002002A1/fr

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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • 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
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention is in the field of evaluation of tumor cells for indications of invasiveness and thus of prognosis of the disease, as a guide for physicians to use in selection or evaluation of treatment.
  • E-cadherin epithelial cell adhesion molecule
  • E-cadherin Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role Cell, 66: 107-119, 1991). In normal physiological conditions, E-cadherin plays an important role in embryonic development, morphogenesis and maintenance of epithelial integrity (see Takeichi, M. The cadherins: cell-cell adhesion molecules controlling animal morphogenesis. Development, 102: 639-655,
  • E-cadherin expression in squamous cell carcinomas of head and neck inverse correlation with tumor dedifferentiation and lymph node metastasis.
  • Cadherin intercellular adhesion molecule in hepatocellular carcinomas loss of E-cadherin expression in an undifferentiated carcinoma.
  • E-cadherin in normal, benign, and malignant tissues of female genital organs Am. J. Clin. Pathol., 98: 76-80, 1992 and Bringuier, P.P., Umbas, R, Schaafsma, H.E., Karthaus, H.F.M., Debruyne, F.M.J., and Schalken, J.A. Decreased E-cadherin immunoreactivity correlates with poor survival in patients with bladder tumors. Cancer Res., 53: 3241-3245, 1993.
  • E-cadherin expression can serve as a prognostic indicator for the biological potential of prostate cancer and thus can function as an important prognostic tool.
  • the other 16 patients were free of progression and have both normal E-cadherin as well as c.-catenin expression.
  • 4 of 13 patients with normal E-cadherin staining show aberrant c.-catenin expression and 2 (50%) were progressed compared with only 22% progression in patients having both normal E-cadherin and ⁇ -catenin expression.
  • the other 19 patients with aberrant E-cadherin and c.-catenin staining have the poorest prognosis.
  • the present invention provides a method for determining invasiveness of an epithelial tumor, which comprises determining a prognostic amount of a prognostic marker selected from the group consisting of E- cadherin and c--catenin in a cell sample obtained from a cell source potentially containing cells of said epithelial tumor and comparing said prognostic amount to a normal amount of said prognostic marker in said cell source, wherein when said prognostic amount is less than said normal amount, said sample is indicative of enhanced invasiveness potential of said ephithelial tumor.
  • the method in particularly advantageous when ⁇ -catenin alone or in combination with E-cadherin is being monitored. These same markers can be followed to monitor treatment efficacy or to screen for new drugs that would be useful for the dedifferentiation of epithelial tumors.
  • Fig. 1 is a photomicrograph showing invasiveness of cells and corresponding labelling of such cells with an antibody to E-cadherin.
  • the different panels of the figure are as follows: a, normal staining, in the well differentiated tumor staining is confined to the cell contacts, b, heterogeneous staining. Positive membranous staining on some of the cells whereas others are negative, c, completely negative staining, none of the tumor cells are stained.
  • E-cadherin immunohistochemistry As a prognostic factor for prostatic cancer patients, and thus the epthelial cancers in general, we analyzed 89 prostate cancer specimens. We found that aberrant E-cadherin expression (heterogeneous, cytoplasmic or negative) was present in 33 % (15 of 46) of the tumors that were clinically found to be organ confined (Tl-2), and in 63% (27 of 43) of the lesions that extended beyond the prostatic capsule (T3-4).
  • E-cadherin expression was not indicative for progression in all of the patients. This may be explained by impaired catenin function through which E-cadherin is anchored to the cytoskeleton. The anchoring function has been studied, but the prognostic relationships have not previously been demonstrated. See, for example, Takeichi, M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science (Washington DC), 251: 1451-1455, 1991, Giroldi, L.A., and Schalken, J.A. Decreased expression of the intercellular adhesion molecule E-cadherin in prostate cancer: biological significance and clinical implications.
  • Cadherin dysfunction in a human cell line possible involvement of loss of c_-Catenin expression in reduced cell-cell adhesiveness. Cancer Res., 52: 5770-5774, 1992.
  • the loss of cell adhesion and the resulting increase in tumor invasiveness resulting from impaired E-cadherin function is a marker of the loss of epithelial integrity, so that monitoring decrease of expression of either E-cadherin or ⁇ -catenin (or preferably both) in epithelial cells provides a valuable technique for following the progression of epithelial cancer.
  • the molecule being detected is referred to as a "prognostic marker, " whether that molecule is E-catenin or ⁇ -cadherin.
  • E-cadherin is an epithelial cell-cell adhesion molecule that is located on the surface of epithelial cells. It is a well known and defined molecule and has been fully sequenced. Numerous publications describing E-cadherin are provided in the background section of this specification.
  • the E-cadherin gene in humans is located on the long arm of chromosome 16 at position 22.1 and is described in a number of publications, including Mansouri et al. Differentiation 38:67-71, 1988.
  • the sequence of the E-cadherin gene and of the cDNA for the expressed polypeptide have been published and are present in several databases, including the EMBL, GENBANK, and DDBJ databases.
  • ⁇ -Catenin is a cytoplasmic epithelial cell polypeptide that acts cooperatively to anchor E-cadherin to the surface of epithelial cells by interactions with the cytoplasmic domain of E-cadherin. It is a well known and defined molecule and has been fully sequenced. Numerous publications describing ⁇ -catenin are provided in the background section of this specification.
  • the ⁇ -catenin gene in humans is located on the long arm of chromosome 5 at position 21-31 and is described in a number of publications (see for example,
  • Two sequences of amino acids are "homologous" if a first sequence at least 10 amino-acid residues in length can be substantially matched on an amino-acid to amino-acid basis with a second sequence, with no more than 20% (two in the case of the minimal length of 10) missing ("gaps") or additional (“inserts") amino acids being allowed in the matching region of the second sequence. More preferably, the gaps and inserts are less than 10%, even more preferably less than 5 %, of the total sequence. Homologies are generally stated in percentages; for example, "at least 80% homology" to a twenty amino acid first sequence would require that the second sequence contain at least 16 identically ordered amino acids, with no more than four gaps or four inserts in the second sequence.
  • Two nucleic acid fragments are "homologous” if they are capable of hybridizing to one another under hybridization conditions described in Maniatis et al . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor NY 1982, pp. 320-323. However, by using the following wash conditions - 2 x SCC, 0.1 % SDS, room temperature twice, 30 minutes each; then
  • homologous sequences can be identified that contain at most about 25-30% basepair mismatches. More preferably, "homologous" nucleic acid sequences contain 15-25 % basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash conditions for identification of clones from gene libraries (or other sources of genetic material), as is well known in the art. The definition of homology above applies generally when percent homology is not stated for homologous sequences.
  • homologous nucleic acid strands contain the idential numerical values (for base pairs rather than amino acid residues) that are stated above for amino acid sequences when percent homologies are stated.
  • a DNA or RNA fragment is "derived from" a prognostic-marker-encoding
  • DNA or RNA sequence if it has the same or substantially the same basepair sequence as a region of the coding sequence for the entire prognostic marker molecule.
  • a probe is typically complementary to such a fragment derived from a marker gene.
  • substantially the same means, when referring to biological activities, that the activities are of the same type although they may differ in degree.
  • substantially the same means that the molecules in question have similar biological properties and preferably have at least 90% homology in amino acid or nucleotide sequences. More preferably, the amino acid sequences are at least 95 % identical. In other uses, "substantially the same” has its ordinary English language meaning.
  • a protein or peptide is "derived from" an prognostic marker molecule if it has the same or substantially the same amino acid sequence as a region of the prognostic marker molecule. Fragments derived from the natural markers are one type of derivative. Such proteins and peptides are useful in the preparation of antibodies specific for the corresponding diagnostic marker. Production of antibodies specific for prognostic markers is discussed elsewhere in this specification. Minor amino acid variations from the natural amino acid sequence of prognostic markers are contemplated as being encompassed by the term prognostic marker; in particular, conservative amino acid replacements are contemplated.
  • an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding properties of the resulting molecule, especially if the replacement does not involve an amino acid at a site involved in induction of an antibody.
  • Whether an amino acid change results in a functional peptide useful for production of an antibody that reacts with an actual prognostic marker can readily be determined by assaying the specific binding properties of the prognostic marker polypeptide derivative or antibody produced.
  • Prognostic markers of the invention for use in the preparation of antibodies and as diagnostic standards can readily be purified from natural sources and from cells genetically modified to produce prognostic marker or polypeptide derivatives or fragments thereof.
  • prognostic markers of the invention preferably means human prognostic markers
  • prognostic markers of mammals e.g. murine, porcine, equine or bovine
  • tissues from such animals can serve as sources of prognostic marker. Homologies are high, in excess of 95 % for various regions of probes and markers derived from markers obtained from different vertebrate species.
  • Crude polypeptide preparations can be purified by numerous techniques, such as by affinity chromatography using a monoclonal antibody specific for the prognostic marker being isolated.
  • monoclonal antibody specific for the prognostic marker being isolated.
  • Such antibodies are commercially available (for example anti E-cadherin from Euro-diagnostica BV, Apeldoorn, the Netherlands or
  • prognostic markers and polypeptide derivatives thereof can be purified by a variety of other widely known protein purification techniques (either alone or in combination) including immunoprecipitation, gel filtration, ion exchange chromatography, chromatofocusing, isoelectric focusing, selective precipitation, electrophoresis, and the like (for example, see Nagafuchi et al., Cell 65:849-857, 1991; Napolitano et al., J. Cell Biol. 113:893-905, 1991; Takeichi, Development, 102:939-655, 1988 and references therein).
  • Fractions isolated during purification procedures can be analyzed for the presence of prognostic marker or polypeptide derivatives of prognostic marker by immunoassays employing prognostic-marker-specific antibodies or prognostic- marker-specific bioassays.
  • GENBANK the genetic information necessary for production of markers by genetic engineering (or for production of probes for analysis) is readily available. This genetic information can be used directly (e.g., for production of probes by automated chemical synthesis) or indirectly (e.g., for isolation of marker genes or fragments thereof from natural sources).
  • Isolation of nucleptide sequences encoding a prognostic marker generally involves creation of either a genomic library prepared from cells encoding prognostic marker or preparation of a cDNA library from RNA isolated from cells expressing prognostic marker. It will generally be preferable to create a cDNA library for isolation of prognostic marker coding nucleotide sequences so as to avoid any possible problems arising from attempts to determine intron/exon borders. Genetic libraries can be made in either eukaryotic or prokaryotic host cells.
  • cloning vectors such as plasmids, cosmids, phage, YACs and the like can be used to generate genetic libraries suitable for the isolation of nucleotide sequences encoding prognostic marker or portions thereof.
  • Useful methods for screening genetic libraries for the presence of prognostic marker nucleotide sequences include the preparation of oligonucleotide probes based on the N-terminus amino acid sequence information from purified prognostic marker or purified internal fragments of purified prognostic marker.
  • oligonucleotide sequences of about 17 base pairs or longer can be prepared by conventional in vitro synthesis techniques so as to correspond to portions of prognostic marker for which the amino acid sequence has been determined by N-terminus analysis.
  • the resultant nucleic acid sequences can be subsequently labeled with radionuclides, enzymes, biotin, fluorescers, or the like, and used as probes for screening genetic libraries.
  • Additional methods of interest for isolating prognostic marker-encoding nucleic acid sequences include screening genetic libraries for the expression of prognostic marker or fragments thereof by means of prognostic marker-specific antibodies, either polyclonal or monoclonal.
  • a particularly preferred technique involves the use of degenerate primers based on partial amino acid sequences of purified prognostic marker or on sequences from known related molecules and the polymerase chain reaction (PCR) to amplify gene segments between the primers.
  • PCR polymerase chain reaction
  • Nucleotide sequences encoding prognostic marker can be obtained from recombinant DNA molecules recovered from prognostic marker genetic library isolates.
  • the nucleotide sequence encoding prognostic marker can be obtained by sequencing the non-vector nucleotide sequences of these recombinant molecules.
  • Nucleotide sequence information can be obtained by employing widely used DNA sequencing protocols, such as Maxim and Gilbert sequencing, dideoxy nucleotide sequencing, and the like. Examples of suitable nucleotide sequencing protocols can be found in Berger and Kimmel, Methods in Enzymologv Vol. 52. Guide to
  • nucleotide sequence information from several recombinant DNA isolates may be combined so as to provide the entire amino acid coding sequence of prognostic marker as well as the nucleotide sequences of introns within the prognostic marker gene, upstream nucleotide sequences, and downstream nucleotide sequences.
  • Nucleotide sequences obtained from sequencing prognostic marker specific genetic library isolates are subjected to analysis in order to identify regions of interest in the prognostic marker gene. These regions of interest include open reading frames, introns, promoter sequences, termination sequences, and the like.
  • Analysis of nucleotide sequence information is preferably performed by computer.
  • Software suitable for analyzing nucleotide sequences for regions of interest is commercially available and includes, for example, DNASIS — (LKB). It is also of interest to use amino acid sequence information obtained from the N-terminus sequencing of purified prognostic marker when analyzing prognostic marker nucleotide sequence information so as to improve the accuracy of the nucleotide sequence analysis.
  • Isolated nucleotide sequences encoding prognostic marker can be used to produce purified prognostic marker or fragments thereof by either recombinant DNA methodology or by in vitro polypeptide synthesis techniques.
  • purified and isolated is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • the term “purified” as used herein preferably means at least 95 % by weight, more preferably at least 99 % by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present).
  • a significant adyantage of producing prognostic marker by recombinant DNA techniques rather than by isolating prognostic marker from natural sources is that equivalent quantities of prognostic marker can be produced by using less starting material than would be required for isolating the binding protein from a natural source.
  • Producing prognostic marker by recombinant techniques also permits prognostic marker to be isolated in the absence of some molecules normally present in cells that naturally produce prognostic marker. Indeed, prognostic marker compositions entirely free of any trace of human protein contaminants can readily be produced since the only human protein produced by the recombinant non- human host is the recombinant prognostic marker. Potential viral agents from natural sources are also avoided. It is also apparent that recombinant DNA techniques can be used to produce prognostic marker polypeptide derivatives that are not found in nature, such as the variations described above.
  • Prognostic marker and polypeptide derivatives of prognostic marker can be expressed by recombinant techniques when a DNA sequence encoding the relevant molecule is functionally inserted into a vector.
  • functionally inserted is meant in proper reading frame and orientation, as is well understood by those skilled in the art.
  • the preferred starting material is a cDNA library isolate encoding prognostic marker rather than a genomic library isolate.
  • the prognostic marker gene will be inserted downstream from a promoter and will be followed by a stop codon, although production as a hybrid protein followed by cleavage may be used, if desired.
  • host-cell-specific sequences improving the production yield of prognostic marker and prognostic marker polypeptide derivatives will be used and appropriate control sequences will be added to the expression vector, such as enhancer sequences, polyadenylation sequences, and ribosome binding sites.
  • the appropriate coding sequence can be expressed in a variety of different expression systems. Only one such system will be described here in detail, as the present invention is not directed to the production or use of E- cadherin or ⁇ -catenin, merely to the detection of these previously known materials and their use in predicting prognosis of epithelial tumor progression.
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase ⁇ to begin RNA synthesis at the correct site.
  • a mammalian promoter will also contain an upstream promoter element, typically located within 100 to 200 bp upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation (Sambrook et al. (1989) "Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories).
  • Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences.
  • Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter.
  • sequences derived from non-viral genes such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone- responsive cells. The presence of an enhancer element (enhancer), combined with the promoter elements described above, will typically increase expression levels.
  • An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000- fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site.
  • Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237: Alberts et al. (1989) Molecular Biology of the Cell. 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they typically have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J.
  • enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:67771 and from human cytomegalovirus [Boshart et al. (1985) Cell 41:5211. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends
  • a DNA molecule may be expressed intracellularly in mammalian cells.
  • a promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
  • foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells.
  • the leader sequence fragment typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • the adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • the 3' terminus of the mature mRNA is formed by site-specific post- transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349: Proudfoot and Whitelaw (1988) "Termination and 3' end processing of eukaryotic
  • RNA In Transcription and splicing (ed. B.D. Hames and D.M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14: 105] . These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells. " In Molecular Cloning: A Laboratory Manual] .
  • genes may be expressed more efficiently when introns (also called intervening sequences) are present.
  • introns also called intervening sequences
  • cDNAs have been effi ⁇ ciently expressed from vectors that lack splicing signals (also called splice donor and acceptor sites) [see e.g., Gothing and Sambrook (1981) Nature 293:620],
  • Introns are intervening noncoding sequences within a coding sequence that contain splice donor and acceptor sites. They are removed by a process called "splicing," following polyadenylation of the primary transcript [Nevins (1983) Annu. Rev. Biochem. 52:441: Green (1986) Annu. Rev. Genet. 20:671: Padgett et al. (1986) Annu. Rev. Biochem. 55: 1119: Krainer and Maniatis (1988) "RNA splicing.”
  • splicing following polyadenylation of the primary transcript
  • Transcription and splicing (ed. B.D. Hames and D.M. Glover)].
  • the above described components comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.
  • Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired.
  • Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria.
  • Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate.
  • plasmids containing the replication systems of papovaviruses such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen.
  • mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus.
  • the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a procaryotic host for cloning and amplification.
  • mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946 and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:10741.
  • E-cadherin and/or ⁇ - catenin can also be produced in other expression systems, such as Baculovirus expression systems, bacterial expression systems, and yeast expression systems.
  • methods for detecting cellular analytes such as prognostic marker proteins of the invention are based on immunoassays. Such techniques are well known and need not be described here in detail. In many cases the assays will be carried out on tissue samples rather than in solution, since the presence of the prognostic markers is associated with cells that adhere to each other. However, it is also possible to assay clusters of cells not found as differentiated tissues (such as ascites tumors) as well as to assays for the presence of the markers (or their expression) by detecting, for example, the markers or mRNA encoding them in solution after the cells of a tissue (or other source) have been dispersed or even lysed. Thus, there is no limit to the cell source being assayed, although it is advantageous to make comparison of prognostic and normal amounts of marker expression using cells from the same cell source.
  • immunohistochemistry is a particularly useful type of assay, as it is possible to directly visualize the presence of the prognostic markers in the cells.
  • a complete description of an immunohistochemical assay is given below (as an example of a qualitative assay). However, it will be recognized that any such example is merely one of a number of variations, of immunohistochemistry. The invention is not limited to any specific assay.
  • Assays based on processes other than immunohistochemistry are also not limited to particular types of assays. Examples include both heterogeneous and homogeneous immunoassay techniques. Both techniques are based on the formation of an immunological complex between the binding protein and a corresponding specific antibody. Heterogeneous assays for prognostic marker typically use a specific monoclonal or polyclonal antibody bound to a solid surface. Sandwich assays are increasingly popular. Homogeneous assays, which are carried out in solution without the presence of a solid phase, can also be used, for example by determining the difference in enzyme activity brought on by binding of free antibody to an enzyme-antigen conjugate. A number of suitable assays are disclosed in U.S. Patent Nos. 3,817,837, 4,006,360, 3,996,345.
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activate carboxyl, hydroxyl, or aldehyde group.
  • a second diagnostic configuration known as a homogeneous assay, antibody binding to an analyte produces some change in the reaction medium which can be directly detected in the medium.
  • homogeneous assays proposed heretofore include (a) spin-labeled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks), (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter.
  • spin-labeled reporters where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks)
  • fluorescent reporters where binding is detected by a change in fluorescence efficiency
  • enzyme reporters where antibody binding effects enzyme/substrate interactions
  • liposome-bound reporters where binding leads to liposome lysis and release of encapsulated reporter.
  • the assay can be either qualitative or quantitative. Since all cells normally contain both of the indicated prognostic markers and since even a single invasive tumor cell can cause deleterious clinical effects, any decrease in the amount or concentration of either of the prognostic markers is clinically significant.
  • a typical qualitative assay can be carried out using immunohistostaining.
  • Indirect immunoperoxidase staining can be carried out as described in detail in Umbas, R., Schalken, J.A., Aalders, T.W. , Carter, B.S., Karthaus, H.F.M., Schaafsma, H.E., Debruyne, F.M.J., and Isaacs, W.B.
  • Expression of the cellular adhesion molecule E-cadherin is reduced or absent in high-grade prostate cancer
  • E-cadherin staining is localized on the membrane, particularly at areas of cell-cell contact, in normal cells.
  • a typical technique for assessing the staining and thus the presence of the prognostic marker is to designate the stained cells as falling into one of three general categories: uniformly positive, uniformly negative, or heterogeneous (mixed populations of positive and negative stained cells).
  • Such a classification has previously been described for other cell types, as described by Schipper et al. (Schipper, J.H., Frixen, U.H., Behrens, J., Unger, A. , Jahnke, K., and Birchmeier, W.
  • Schipper et al. Schopper, J.H., Frixen, U.H., Behrens, J., Unger, A. , Jahnke, K., and Birchmeier, W.
  • E-cadherin expression in squamous cell carcinomas of head and neck inverse correlation with tumor dedifferentiation
  • a predetermined range of concentrations for the same cell source in the normal population is typically obtained by using the same assay technique that will be used in the application of the method to an individual being tested, in order to ensure the highest correlation.
  • sufficient measurements are made in a normal population to produce a statistically significant range of normal values for the value to which a comparison will be made.
  • the minimum concentration (or other amount) indicative of increased invasiveness is considered to be (for example) 1, 2, 3, or 4 standard deviations below the mean prognostic marker concentration for the normal population, for any given cell source.
  • one standard deviation would encompass about 68% of normal samples; that is, 32% of normal samples would be expected to fall outside the lower and upper limits set by one standard deviation from the mean (16% would thus be expected to be below the selection limit).
  • one standard deviation above the normal mean would not be used for definitive analysis for increased invasiveness, as it would include too many false positives.
  • one standard deviation is appropriate for an assay that is desired to sweep in for further evaluation all possible candidates who might be predisposed to metastasis, as in a patient with a personal or family history of metastasis.
  • Two standard deviations from the mean would encompass about 95 % of normal samples; three standard deviations, about 99%; four standard deviations, more than 99%. These levels are more appropriate generally, especially for samples from a cell source or patient that show a high coefficient of variance.
  • the limit of the indication of invasiveness (lower limit of the normal range) in standard deviations. Any other system that can be used to provide a statistically significant indication of invasiveness can be used.
  • the limit can be set to be a concentration (or other measure) that is at least as high as the 95th percentile concentration for normal patients for the same cell source.
  • Prognostic-mark ⁇ r-specific binding molecules include polypeptides such as antibodies that are specific for the prognostic marker polypeptide containing the naturally occurring prognostic marker amino acid sequence.
  • specific binding polypeptide is intended polypeptides that bind with a prognostic marker and which have a measurably higher binding affinity for the target polypeptide, i.e., prognostic marker and polypeptide derivatives of prognostic marker, than for other polypeptides tested for binding. Higher affinity by a factor of 10 is preferred, more preferably a factor of 100.
  • Binding affinity for antibodies refers to a single binding event (i.e., monovalent binding of an antibody molecule). Specific binding by antibodies also means that binding takes place at the normal binding site of the antibody (i.e., at the end of the arms in the variable region).
  • Prognostic markers both glycosylated and unglycosylated (E-cadherin is normally glycosylated; ⁇ -catenin is not), or polypeptide derivatives thereof, may be used for producing antibodies, either monoclonal or polyclonal, specific to prognostic marker.
  • polypeptide derivatives polypeptides differing in length from natural prognostic marker and containing five or more amino acids from the prognostic marker in the same primary order as found in the prognostic marker as obtained from a natural source.
  • Polypeptide molecules having substantially the same amino acid sequence as a prognostic marker but possessing minor amino acid substitutions that do not substantially affect the ability of the prognostic marker polypeptide derivatives to interact with prognostic marker- specific molecules, such as antibodies, are within the definition of prognostic marker.
  • Derivatives include glycosylated forms, aggregative conjugates with other prognostic marker molecules, and covalent conjugates with unrelated chemical moieties.
  • Covalent derivatives are prepared by linkage of functionalities to groups which are found in the prognostic marker amino acid chain or at the N- or C- terminal residue by means known in the art.
  • Antibodies specific for a prognostic marker are produced by immunizing an appropriate vertebrate host, e.g., rabbit, with purified prognostic marker or polypeptide derivatives of prognostic marker, by themselves or in conjunction with a conventional adjuvant. Usually, two or more immunizations will be involved, and blood or spleen will be harvested a few days after the last injection. For polyclonal antisera, the immunoglobulins can be precipitated, isolated and purified by a variety of standard techniques, including affinity purification using a prognostic marker attached to a solid surface, such as a gel or beads in an affinity column.
  • the splenocytes normally will be fused with an immortalized lymphocyte, e.g., a myeloid cell line, under selective conditions for hybridoma formation.
  • an immortalized lymphocyte e.g., a myeloid cell line
  • spleen or lymphocytes from an immunized animal can be removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art.
  • a human lymphocyte donor is selected.
  • Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human- human hybridomas.
  • Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies.
  • Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity. Techniques for producing antibodies are well known in the literature and are exemplified by the publication Antibodies: A Laboratory Manual
  • the concentration of the prognostic marker in the cell sample being assayed is correlated with a standard value to determine when increased invasiveness of cells is present.
  • the standard is usually (1) a predetermined range of prognostic marker concentrations (or other units of amount) for the same cell source in the general population as that being assayed, or (2) a previously measured prognostic marker amount or concentration from the same cell source.
  • a lower measured concentration of prognostic marker relative to the standard value is an indication of increased invasiveness and thus poorer likely prognosis of the patient. Treatment can then be initiated or changed by the attending physician based on this information.
  • the analyte can be a nucleotide sequence which hybridizes with a probe comprising a sequence of (usually) at least about 16 consecutive nucleotides, usually 30 to 200 nucleotides, up to substantially the full sequence of the sequences shown above (cDNA sequences).
  • the analyte can be RNA or cDNA for full-length probes or shorter genetic materials for probes that are capable of binding to the intron regions of genomic
  • a positive result is generally characterized as identifying a genetic material comprising a sequence at least about 70% homologous to a sequence of at least 12 consecutive nucleotides, preferably 20 consecutive nucleotides, more preferable 40 consecutive nucleotides, of the sequences described herein, usually at least about 80%, preferably 90%, more preferably 95 % , homologous to at least about 60 consecutive nucleotides within the sequences, and may comprise a sequence substantially homologous to the full-length sequences or some part thereof.
  • the probe can contain a detectable label.
  • Probes that are particularly useful for detecting presence of the markers are based on conserved regions of prognostic marker proteins that have been screened (either by computer analysis of sequence databases or by hybridization screening against marker-deficient cells of the type being assayed) for the absence of non-marker hybridization under the conditions being used in the assay.
  • One method for amplification of target nucleic acids, for later analysis by hybridization assays or as part of a detection scheme by itself, is known as the polymerase chain reaction or PCR technique.
  • the PCR technique can be applied to detecting prognostic marker of the invention in suspected samples using oligonucleotide primers spaced apart from each other and based on the published genetic sequences of the prognostic markers.
  • the primers are complementary to opposite strands of a double stranded DNA molecule and are typically separated by from about 50 to 450 nt or more (usually not more than 2000 nt).
  • This method entails preparing the specific oligonucleotide primers and then repeated cycles of target DNA denaturation, primer binding, and extension with a DNA polymerase to obtain DNA fragments of the expected length based on the primer spacing.
  • Extension products generated from one primer serve as additional target sequences for the other primer.
  • the degree of amplification of a target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2n where n is the number of cycles. Given that the average efficiency per cycle ranges from about 65 % to 85% , 25 cycles produce from 0.3 to
  • the starting material for PCT analysis can be either DNA or RNA.
  • Messenger RNA is a preferred target, since such mRNA is present when active expression of a prognostic marker is taking place.
  • the PCR method is described in a number of publications, including Saiki et al., Science (1985) 230:1350-1354; Saiki et al., Nature (1986) 324:163-166; and Scharf et al., Science (1986) 233:1076-1078. Also see U.S. Patent Nos. 4,683,194; 4,683,195; and 4,683,202.
  • the invention includes a specific diagnostic method for determination of prognostic marker, based on selective amplification of prognostic marker-encoding DNA fragments.
  • This method employs a pair of single-strand primers derived from non-homologous regions of opposite strands of a DNA duplex fragment selected from the published prognostic marker. These "primer fragments," which form one aspect of the invention, are prepared from prognostic marker fragments such as described above.
  • the method follows the process for amplifying selected nucleic acid sequences as disclosed in U.S. Patent No. 4,683,202, as discussed above.
  • the action to be taken after determining a prognostic marker level indicative of invasiveness is best selected by an attending physician or veterinarian after collecting data from several samples.
  • quantitatively abnormal expression of a prognostic marker would typically suggest a more aggressive treatment than that currently being used. If metastatic disease is already established, the clinician can adjust patient expectations of prognosis according to the therapy options available, or can switch options, if no benefit is seen from the current therapy.
  • the method of the invention can be used to follow the prognosis of any epithelial tumor, including but not limited to colon, gastric, cervical, breast, prostate, bladder, head and neck, basal cell, thyroid, and meningioma tumors of epithelial origin.
  • a previously measured prognostic marker concentration of the same cell source of the same patient can be used as a standard for comparison. In this case, what is being determined is usually the rate of increase or decrease in prognostic marker concentration as an indication of the progression of the disease or the success of the treatment.
  • a positive assay i.e., indication of decreased invasiveness
  • a patient who previously showed only 50% positive marker cells in a tissue sample might show 75% positive after a particular treatment (e.g., chemotherapy with a given drug, where previous chemotherapy with different drugs did not show improvement in presence of these prognostic markers).
  • a particular treatment e.g., chemotherapy with a given drug, where previous chemotherapy with different drugs did not show improvement in presence of these prognostic markers.
  • the selection of a particular change as indicative of a change in treatment is best selected by the attending physician for the particular reason desired.
  • Increases in prognostic marker concentration that meet the standards of this paragraph and additionally reach the essential homogeneous positive patterns of markers found in normal populations of patients are particularly indicative of appropriate treatment.
  • Compounds can be evaluated for potential redifferentiation activity for epithelial cells by adding a test compound to a cell composition deficient in expression of E-cadherin or ⁇ -catenin, monitoring the cell composition for increased expression of the appropriate marker, and selecting the test compound for use or additional testing as a redifferentiation compound if the cell composition shows increased expression of the marker. Any of the techniques described above can be applied to the monitoring process.
  • the epithelial cell line deficient in marker expression can be selected on the basis of prior knowledge of marker expression or can readily be selected from epithelial cell tumor lines (e.g., obtainable from the
  • Bone scan was not done in one patient with T4 tumor.
  • Treatment and/or radiotherapy in 4 cases.
  • TUR-P Transurethral resection of the prostate. Including 3 patients with pre-operative adjuvant hormonal therapy.
  • Transurethral resection or radical prostatectomy specimens were snap frozen. Sections of 4-6 ⁇ m thickness were cut on a cryostat, air-dried, and stored at -20°C until use. One section from each patient was stained with hematoxylin and eosin to assess the histopathological grade according to Gleason (Gleason, D.F. Histologic grading and clinical staging of prostatic carcinoma. In: M. Tannenbaum (ed.), Urologic Pathology: The Prostate, pp. 171-197. Philadelphia: Lea and Febiger, 1977). In the group of patients described in Part 1 of Example 1, tumor grade ranged from Gleason score 4 to 10.
  • Indirect immunoperoxidase staining was performed as described in Umbas, et al. 1992, using either anti E-cadherin (Eurodiagnostica BV, Apeldoorn, The Netherlands) or HECD-1 (Takara, Berkeley, U.S.A.) monoclonal antibodies. E-cadherin staining is localized on the membrane, particularly at areas of cell-cell contact. To assess the sjtaining we used the following criteria: uniformly positive, uniformly negative or heterogeneous (mixed populations of positive and negative stained cells) as described by Schipper et al.
  • cytoplasmic staining Besides positive or negative staining, some tumors showed a cytoplasmic staining, which was also considered to be abnormal and included in the criteria for heterogeneous staining. Uniformly positive staining patterns were regarded as normal while uniformly negative and heterogeneous stainings were considered as aberrant expression.
  • Part 5 Analysis results Eighty-nine snap frozen prostate cancer specimens were stained with the anti-E-cadherin monoclonal antibodies anti-E-cadherin or HECD-1 and scored as described in Part 3 of Example 1.
  • the scoring system is based on the biological functional relation between loss of E-cadherin expression and invasiveness, i.e., the presence of a negative subpopulation is considered to have important biological significance.
  • Figure la an example of normal expression can be seen with uniformly positive staining on the membrane at cell-cell contacts. Abnormal patterns comprise partially positive and partially negative stainings on the membrane or a cytoplasmic staining (Fig. lb). Occasionally, no E-cadherin expression at all was found in the entire tumor area evaluated (Fig. lc). Both of the latter staining patterns were regarded as aberrant expression of this molecule.
  • E-cadherin expression was significantly correlated with aberrant E-cadherin expression (Table 2, p ⁇ 0.001).
  • E-cadherin expression was normal in 68 % and aberrant in 32%.
  • E-cadherin expression was normal in 24% and aberrant in 76% of the patients that presented with metastases (Table 2).
  • Table 2 Relationship of E-cadherin expression to tumor grade, clinical stage and metastases.
  • TUR-P transurethral resection of the prostate
  • Treatment options were radical prostatectomy for early stage and hormonal treatment for advanced disease.
  • follow-up was done by measuring prostate specific antigen level, and bone scan was performed to detect metastatic lesions.
  • Part 2 Surgical specimens.
  • Part 3 Antibodies.
  • Immunohistochemistry was performed by using indirect method as described previously (Umbas 1992) except that we used biotinylated (Amersham) anti-mouse Ig for E-cadherin and anti-rat Ig for ⁇ -catenin staining as the second antibody and incubated with avidin-biotin-peroxidase complex (Vectastain ABC kit; Vector Laboratories Inc., Burlingame, CA) before incubation with diaminobenzidine 0.6 mg/ml in 0.65% Imidazol/Phosphate-buffered saline.
  • biotinylated (Amersham) anti-mouse Ig for E-cadherin and anti-rat Ig for ⁇ -catenin staining as the second antibody and incubated with avidin-biotin-peroxidase complex (Vectastain ABC kit; Vector Laboratories Inc., Burlingame, CA) before incubation with diaminobenzidine 0.6 mg/
  • E-cadherin and ⁇ -catenin staining patterns were scored as described in Example 1 ; uniformly positive staining was regarded as normal, while heterogeneous, uniformly negative, and cytoplasmic stainings were scored as aberrant staining.
  • Example 1 there was significant correlation between E- cadherin expression and tumor grade, tumor stage, survival of the advanced stage patients, and progression free interval in patients with low stage disease treated by radical prostatectomy.
  • Table 6 Relationship of ⁇ -catenin expression to tumor grade.
  • Example 3 Evaluation of redifferentiation drugs using E-cadherin expression as a diagnostic
  • Examples 1 and 2 suggested to us the potential for using these prognosis markers in model systems used to evaluate candidate drugs for their ability to induce reexpression of the marker and thus dedifferentiation of tumors.

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Abstract

Méthode permettant de déterminer le pouvoir envahissant relatif d'une tumeur épithéliale, consistant à déterminer la quantité pronostique d'un marqueur pronostique sélectionné dans le groupe composé de E-cadhérine et α-caténine dans un échantillon de cellules prélevé sur une source cellulaire susceptible de contenir des cellules de la tumeur épithéliale, et à comparer la quantité pronostique à une quantité normale de marqueur pronostique dans la source cellulaire. Si la quantité pronostique est inférieure à la quantité normale, l'échantillon indique un potentiel accru du pouvoir envahissant des cellules de la tumeur épithéliale. On peut utiliser ce procédé également pour contrôler l'efficacité de différentes thérapies et pour déceler des composés candidats pouvant être utilisés dans le traitement des tumeurs épithéliales.
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Cited By (6)

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WO1997036535A3 (fr) * 1996-03-29 1998-03-26 Univ Texas Biomarqueurs servant a effectuer la detection, le diagnostic et le pronostic du cancer de la prostate
WO2001002860A1 (fr) * 1999-07-01 2001-01-11 Consejo Superior De Investigaciones Cientificas Snail, nouveau marqueur de progression tumorale et proteine diana de nouveaux composes antitumoraux
WO2001047954A3 (fr) * 1999-12-23 2001-11-29 Vlaams Interuniv Inst Biotech Nouveaux adnc codant des proteines de fixation a catenine et possedant une fonction de signalisation et/ou de regulation genique
WO2000060119A3 (fr) * 1999-04-01 2002-01-10 Us Gov Health & Human Serv Technique permettant de detecter les cellules cancereuses
WO2002004636A1 (fr) * 2000-07-12 2002-01-17 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Nouvelle alpha-catenine exprimee dans le coeur et les testicules
WO2005083445A3 (fr) * 2004-02-26 2005-11-24 Procter & Gamble Methodes de determination des bienfaits relatifs et/ou d'evaluation de changements quantitatifs causes par des produits sur des tissus epitheliaux

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JPH0630785A (ja) * 1992-07-10 1994-02-08 Eisai Co Ltd αカテニンに対するモノクローナル抗体
WO1994011401A1 (fr) * 1992-11-17 1994-05-26 Yale University Homologue humain du gene de la cadherine e et procedes d'utilisation

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WO1992017608A1 (fr) * 1991-03-28 1992-10-15 Walter Birchmeier Procede de detection de la differentiation et de l'invasivite de cellules de carcinomes
JPH0630785A (ja) * 1992-07-10 1994-02-08 Eisai Co Ltd αカテニンに対するモノクローナル抗体
WO1994011401A1 (fr) * 1992-11-17 1994-05-26 Yale University Homologue humain du gene de la cadherine e et procedes d'utilisation

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5861248A (en) * 1996-03-29 1999-01-19 Urocor, Inc. Biomarkers for detection of prostate cancer
US6090559A (en) * 1996-03-29 2000-07-18 Urocor, Inc. Biomarkers for the detection of prostate cancer
WO1997036535A3 (fr) * 1996-03-29 1998-03-26 Univ Texas Biomarqueurs servant a effectuer la detection, le diagnostic et le pronostic du cancer de la prostate
WO2000060119A3 (fr) * 1999-04-01 2002-01-10 Us Gov Health & Human Serv Technique permettant de detecter les cellules cancereuses
US7482126B2 (en) 1999-07-01 2009-01-27 Consejo Superior De Investigacoines Cientificas Snail, a new marker for tumour invasion and target protein of new antitumoral compounds
WO2001002860A1 (fr) * 1999-07-01 2001-01-11 Consejo Superior De Investigaciones Cientificas Snail, nouveau marqueur de progression tumorale et proteine diana de nouveaux composes antitumoraux
ES2161612A1 (es) * 1999-07-01 2001-12-01 Consejo Superior Investigacion Procedimiento para identificar un compuesto que inhiba la funcion represora de snail.
ES2161655A1 (es) * 1999-07-01 2001-12-01 Consejo Superior Investigacion Procedimiento para determinar la capacidad invasiva y metastasica de un tumor epitelial mediante el uso de snail.
US7794930B2 (en) 1999-07-01 2010-09-14 Consejo Superior De Investigaciones Cientificas Snail, a new marker for tumour invasion and target protein of new antitumoral compounds
US7482159B2 (en) 1999-07-01 2009-01-27 Consejo Superior De Investigaciones Cientificas Snail, a new marker for tumour invasion and target protein of new antitumoral compounds
WO2001047954A3 (fr) * 1999-12-23 2001-11-29 Vlaams Interuniv Inst Biotech Nouveaux adnc codant des proteines de fixation a catenine et possedant une fonction de signalisation et/ou de regulation genique
US7341866B2 (en) 2000-07-12 2008-03-11 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw α-catenin expressed in heart and testis
WO2002004636A1 (fr) * 2000-07-12 2002-01-17 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Nouvelle alpha-catenine exprimee dans le coeur et les testicules
US7229778B2 (en) 2004-02-26 2007-06-12 The Procter & Gamble Company Methods for determining the relative benefits and/or evaluating quantitative changes of products on epithelial tissue
WO2005083445A3 (fr) * 2004-02-26 2005-11-24 Procter & Gamble Methodes de determination des bienfaits relatifs et/ou d'evaluation de changements quantitatifs causes par des produits sur des tissus epitheliaux
US7763419B2 (en) 2004-02-26 2010-07-27 The Procter & Gamble Company Methods for determining the relative benefits and/or evaluating quantitative changes of products on epithelial tissue
US7767389B2 (en) 2004-02-26 2010-08-03 The Procter & Gamble Company Methods for determining the relative benefits and/or evaluating quantitative changes of products on epithelial tissue
US7771925B2 (en) 2004-02-26 2010-08-10 The Procter & Gamble Company Methods for determining the relative benefits and/or evaluating quantitative changes of products on epithelial tissue
US7771924B2 (en) 2004-02-26 2010-08-10 The Procter & Gamble Company Methods for determining the relative benefits and/or evaluating quantitative changes of products on epithelial tissue

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