WO1994000764A1 - Proto-oncogene met et procede de prediction de l'evolution du cancer du sein - Google Patents

Proto-oncogene met et procede de prediction de l'evolution du cancer du sein Download PDF

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WO1994000764A1
WO1994000764A1 PCT/US1993/005952 US9305952W WO9400764A1 WO 1994000764 A1 WO1994000764 A1 WO 1994000764A1 US 9305952 W US9305952 W US 9305952W WO 9400764 A1 WO9400764 A1 WO 9400764A1
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Prior art keywords
met
tissue
tumor
breast
normal
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PCT/US1993/005952
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English (en)
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Ilan Tsarfaty
James H. Resau
Iafa Keydar
Donna Faletto
George F. Vande Woude
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THE UNITED STATES OF AMERICA, represented by THE SECRETARY OF HEALTH AND HUMAN SERVICES
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Priority claimed from US07/903,588 external-priority patent/US6673559B1/en
Application filed by THE UNITED STATES OF AMERICA, represented by THE SECRETARY OF HEALTH AND HUMAN SERVICES filed Critical THE UNITED STATES OF AMERICA, represented by THE SECRETARY OF HEALTH AND HUMAN SERVICES
Priority to AU46452/93A priority Critical patent/AU4645293A/en
Publication of WO1994000764A1 publication Critical patent/WO1994000764A1/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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development

Definitions

  • Met met protooncogene product
  • Met protein a member of the family of tyrosine kinase growth factor receptors
  • Met is synthesized as a glycosylated 170-kD precursor and cleaved in the external (ligand binding) domain to yield a mature disulfide-linked ⁇ - (50-kD) , ⁇ - (140-kD) heterodi er (C.S. Cooper et al . , Nature 311: 29 (1984); S.
  • the Met receptor is expressed in a wide variety of tissue and cell types, but the highest levels are found in epithelial cells (M.F. Di Renzo et al . , Oncogene 6: 1997-2003 (1991); A. Iyer et al . , Cell Growth & Diff . 1: 87 (1990)).
  • Hepatocyte growth factor was first purified from human and rabbit plasma and rat platelets on the basis of its ability to stimulate mitogenesis of rat hepatocytes (E. Gohoda et al., J. Clin . Invest . 81: 414 (1988); R. Zarnegar and G. Michalopoulos, Cancer Res . 49: 3314 (1989); T. Naka ura et al. FEBS Lett . 224: 311 (1987)) .
  • HGF may act as a humoral factor promoting liver regeneration after partial hepatectomy or liver injury (G.K. Michalopoulos, FASEB J . 4: 176 (1990)).
  • HGF may also act as a paracrine mediator of proliferation for a broad spectrum of cell types.
  • Molecular cloning of HGF revealed a remarkable structural homology to plasminogen and related serine proteases (J.S. Rubin et al., Proc . Natl . Acad. Sci . U.S.A. 415 (1990); T. Nakamura et al., Nature 342: 440 (1989); K. Miyazawa et al., Biophys . Res . Comm . 163: 967 (1989)) .
  • HGF is structurally related to the family of serine proteases that includes plasminogen, prothrombin, urokinase, and tissue plasminogen activator (J.S. Rubin et al., Proc . Natl . Acad . Sci . U.S .A . 88: 415 (1990)); T. Nakamura et al. , Nature 342: 440 (1989)).
  • HGF includes a variant of HGF previously characterized as a broad-spectrum mitogen called plasminogen like growth factor (PLGF) .
  • PLGF plasminogen like growth factor
  • Patent Application Serial Number 07/642,971 describes the complex comprising HGF and met protooncogene protein and identifies the met protooncogene as the receptor for HGF.
  • Scatter factor (SF) originally had been considered to be related to but different from HGF, SF being associated with cell motogenicity (motility) , and HGF being associated with cell mitogenicity (growth) .
  • HGF/SF HGF/SF
  • HGF/SF The scattering response of HGF/SF establishes that this factor is responsible for cell motility and differentiation (G.F. Vandewoude Japan . J. Can . Res . 83: cover (1992)).
  • MDCK cells grown in collagen gels in the presence of HGF/SF form bracing tubules, suggesting that the three-dimensional geometry of cell-substrate interactions directs MDCK cells to organize into tubules in response to HGF/SF (R. Montesano et al . , Cell 67: 901 (1991)). It is known that mammary gland epithelia undergo developmental changes during pregnancy to become secretory.
  • mammary epithelial cells can regain their differentiated phenotype only when appropriate hormonal and substratum conditions are provided (M.J. Bissell et al . , The Mammary Gland, M. Neville and C. Daniel, Eds. (Plenum Press Publishing Corp., New York, pp. 97-146 (1987); CH. Streuli et al . , J . Cell Biol . 115: 1383 (1991)).
  • a need existed to determine whether there was met expression in normal breast duct cells and to determine the role that HGF/SF and met play in the differentiation of ductal epithelium in the mammary gland.
  • Bieche et al used the c-met proto-oncogene probe, which detects sequences on chromosome 7q31, to analyse tumor and blood leucocyte DNA samples from 245 patients with primary breast cancers.
  • the pmet H polymorphic probe detected a high frequency of allele loss (40-50%) among the 121 informative (heterozygous) patients.
  • one object of the present invention is to provide a method for predicting the progression of breast cancer based upon the presence of Met protein in normal and breast cancer tissue. Such a method would be advantageous in the management of breast cancer therapy, used either alone or in conjunction with other prognostic features such as tumor size, histo- and cytopathological grade and lymph node or steroid receptor status.
  • the present invention relates to a method for predicting breast tumor progression.
  • This method involves contacting a section from a breast tumor with an antibody reagent specific for Met protein under antibody binding conditions, wherein the section contains normal breast duct tissue and tumor tissue; determining the binding of the reagent to Met protein in the normal tissue and the tumor tissue; and comparing the binding of the reagent to met protein in the normal tissue with the binding in the tumor tissue; wherein, greater binding of the reagent to normal than to tumor tissue indicates a high likelihood of tumor metastasis.
  • Figure 1 shows the expression of Met in cells bordering lumen-like structures of the breast cancer cell line T47D.
  • T47D cells were treated with Bouin's fixative, embedded in paraffin, serial sectioned, and stained by indirect immunofluorescence. Cells were analyzed using confocal laser scanning microscopy (CLSM) .
  • Figure IA shows a Nomarski image of the lumen-like structures in T47D cells in culture.
  • Figure IB shows immunofluorescent staining of the same region as in Figure IA with Met C28 antibody.
  • Figures IA and IB are X2.45 magnification of the lower right hand portion of Figures IA and IB. Overall magnification is X490.
  • Figure 1C through IE show quantitative determination of the Met-specific immunofluorescence.
  • the histograms show the distribution of fluorescence of the bracketed areas.
  • Figure 1C specifically shows the fluorescence in the apical region of the lumen for the right lumen.
  • Figure ID specifically shows the fluorescence in the apical region of the lumen for the upper left lumen.
  • Figure IE specifically shows the fluorescence in the apical region of the lumen for an area adjacent to the upper left lumen.
  • FIG. 2 shows subcellular localization of Met in T47D cells.
  • Immunoelectron microscopy was performed using secondary antibody labeled with 10-nm colloidal gold particles (magnification 30,OOOX) (For postembedding immunoelectron microscopy, fixed cells were embedded in L.R. gold resin (Electron Microscopy Science) at -25°C, sectioned with an LKB Nova Ultratome, and picked up with Formvar-coated 200 mesh gold grids. The grids were washed three times in PBS 10 in and incubated in 1% BSA (in PBS) for 2 hours and in rabbin anti-human C28 Met antibody (diluted 1:50 in 1% BSA) at room temperature for 1 hour.
  • 1% BSA in PBS
  • rabbin anti-human C28 Met antibody diluted 1:50 in 1% BSA
  • Controls were incubated either in the presence of C28 competing peptide or in the absence of the primary antibody.
  • the grids were washed again in PBS, incubated in RPM11640 medium for 20 min in 1% BSA as above, and reacted with goat anti-rabbit IgG gold (10-nm diameter; 1:10 diluted in 1% BSA; Amersham) at room temperature for 1 hour.
  • the grids were finally washed in PBS and distilled water and stained with uranyl acetate and lead citrate. The sections were observed and photographed using a Philips EM 410 electron microscope.).
  • Met is localized in microvilli that protrude into lumen (L) and could account for the apical staining observed in cells bordering the lumen (A and B) .
  • Figure 3 shows expression of Met in breast tissue biopsies.
  • a paraffin-embedded section of biopsy from breast adenocarcino a was subjected to indirect immunofluorescence staining with C28 antibody or monoclonal antibody 4G10.
  • the cells and immunofluorescence were analyzed by CLSM.
  • Figure 3A shows immunofluorescence of normal mammary duct, performed with C28. Magnification X250.
  • Figure 3B shows immunofluorescence of normal mammary duct, performed with anti-P-Tyr. Magnification X250.
  • Figure 3C shows immunofluorescence of normal mammary duct, performed with C28. Magnification X250.
  • Figures 4A-F relate to expression of Met in the mouse embryonal digestive tract. Eleven-day-old BALB/c mouse embryos were fixed, embedded in paraffin, and subjected to immunofluorescence staining with SP260 and 4G10 antibodies. Magnification is X220.
  • Figure 4A shows apical staining of the lumen of the digestive tract with SP260.
  • Figure 4B shows staining in the absence of the primary SP260 antibody.
  • Figure 4C shows staining in the presence of SP260 competing peptide.
  • Figure 4D shows apical staining of the lumen of the digestive tract with SP260.
  • Figure 4E shows immunofluorescence with 4G10.
  • Figure 4F shows an analysis of the overlap between the Met and the anti-P-Tyr staining by a confocal image processing program.
  • Figures 5A-F relates to HGF/SF induction of lumen formation in S 480 and HT29 human colon carcinoma cell lines.
  • the cells were treated with the specified concentration of HGF/SF when they were -70% confluent.
  • Figure 5A shows S 480 cells grown for 18 hours in complete medium without HGF/SF.
  • Figure 5B shows S 480 cells grown for 18 hours in complete medium with HGF/SF ⁇ -.' ⁇ :. 1 ng/ml.
  • Figure 5C shows SW480 c-sils grown for 18 hours in complete medium with HGF/SF at 5 ng/ml.
  • Figure 5D shows S 480 cells grown for 18 hours in complete medium with HGF/SF at 10 ng/ml.
  • Figure 5E shows SW480 cells grown for 18 hours in complete medium with HGF/SF at 100 ng/ml.
  • Figure 6 shows the influence of HGF/SF concentration on lumen formation. The number of cells forming lumen structures was calculated for each HGF/SF concentration.
  • Met protein and RNA products are prognostic in evaluating tumor progression towards metastasis in human breast cancer.
  • the expression of met in certain organs is associated with epithelial cell differentiation in tumor cells. More specifically, applicants have discovered that expression of the met protooncogene product in normal tissues is associated with the formation of highly ordered, fully differentiated lumen structures and that the ligand for Met, HGF, plays a key role in inducing the formation of these structures. It also has been discovered that while met expression is very high in cells that form normal breast ducts, expression of met in adjacent, poorly differentiated, cancer tissue is markedly reduced. This information has led to the further discovery that reduced met expression correlates with poor prognosis in human breast cancer.
  • the method of the present invention involves comparing the amount of Met in normal breast tissue with the amount of Met in tumor tissue. This is accomplished by determining one or more of met DNA abundance, met mRNA abundance, or Met protein abundance in normal breast tissue and breast tumor tissue from the same patient. The detection of a higher abundance of met DNA, met mRNA or Met protein in normal tissue than in tumor tissue is indicative of a poor prognosis.
  • the method involves contacting a sample of breast tissue containing both normal and tumor cells with an antibody reagent specific for Met protein, under antibody binding conditions. Greater binding of the reagent to normal than to tumor tissue indicates a high likelihood of tumor metastasis and a poor prognosis.
  • the breast tissue sample is a "section,” i.e., a histological section obtained from a surgical or biopsy procedure, according to techniques well known in the art.
  • the term "contacting" is intended to encompass any technique which permits the antibody reagent to bind with Met protein.
  • antibody reagents whole antibodies and parts thereof, either alone or conjugated with other moieties.
  • Antibodies include polyclonal antibodies, monoclonal antibodies, and single chain antibodies.
  • Antibody fragments are those the bind the Met protein, including Fab and F(ab) 2 fragments, inter alia .
  • the antibody reagents may comprise antibodies made in animals or by recombinant DNA techniques.
  • the antibody reagents include antibody and antibody fragments conjugated to, among other moieties, detectable labels, such as enzymatic labels and fluorescent labels. Other useful labels which may be comprised by the antibody reagents include radionuclides.
  • Antibody binding conditions generally are well known in the art and, for the most part will include neutral pH, moderate salt, temperatures between 2-3 C and 37° C, incubation times between several minutes and overnight or longer. Preferred conditions include those described by M. Gonzatti-Haces et al . , Proc . Natl . Acad . Sci . USA 85: 21 (1988) for a rabbit antibody prepared against the extracellular domain of the Met peptide, those of M. Park et al . , Proc . Natl . Acad . Sci . USA 84: 6379 (1987) for a rabbit anti-mouse Met and the conditions described by D.K. Morrison et al .
  • greater binding is readily understood to be a relative term which derives meaning by comparing the amount of bound Met in normal with the amount of bound Met in tumor cells. An 80- to 150-fold or greater reduction in Met in tumor cells is considered predictive of a poor prognosis. hether a prognosis is good or poor depends upon the likelihood of metastasis. A "high likelihood" of tumor metastasis means that there is about an 80-90% chance of tumor metastasis within about two years.
  • a histological tumor breast biopsy section containing both normal duct tissue and adjacent tumor tissue is embedded in paraffin and blocked for about 10 minutes using a commercially available immunostaining blocking reagent.
  • a primary antibody is then incubated with the tissue.
  • the preferred primary antibody is selected from the group consisting of C28 (rabbit anti-human Met) , C200 (rabbit antibody against the extracellular domain of the Met peptide) , SP260 (rabbit anti-mouse Met) , 4G10 (mouse monoclonal antibody to phosphorylated tyrosine (P-Tyr) and the 19S monoclonal (previously described in U.S. patent application serial no. 07/457,556) .
  • the tissue is then further incubated with a secondary antibody coupled to a fluorochrome.
  • the secondary antibody may be donkey anti-rabbit coupled to phycoerythrin or donkey anti-mouse coupled to fluorescein isothiocyanate.
  • Antibody binding is determined by examining the fluorochrome labeled cells with a conventional light or confocal laser scanning microscope, such as the Zeiss laser scan microscope.
  • the fluorescent intensity is then quantitated using methods known in the art.
  • the relative fluorescence intensity (the intensity of the normal compared with tumor tissue) is quantitated with an Indec Laser Scan Microscope Image Processor Option.
  • photographs may be taken to document visualized tissue.
  • the abundance of met mRNA or met DNA in a breast tissue sample may be detected by in situ hybridization using met sequence specific probes, or by hybridization of met sequence specific probes to mRNA or DNA from normal and tumor tissue.
  • Probe complementary to met are prepared by methods known in the art and allowed to hybridize to mRNA or DNA within a section of a tissue sample (either embedded by standard techniques, e.g. paraffin, or otherwise preserved) . Unhybridized probe are removed by nuclease. Hybridization can be detected by autoradiography or other methods. The intensity of hybridization reflects the amount of met within the cells of the tissue. The observation of tumor tissue containing reduced levels of met relative to levels in normal tissue, indicates a poor prognosis.
  • the polymerase chain reaction is used to detect met DNA or mRNA in a breast tissue sample.
  • PCR polymerase chain reaction
  • a pair of met sequence specific primers is employed, which hybridize to opposite strands of the met gene at offset positions on the double helix.
  • the primers provide initiation points for DNA synthesis.
  • NTPs nucleotide triphosphates
  • other necessary co-factors which are all well known to the art
  • new DNA strands are synthesized complementary to the templates which hybridized with the primers.
  • Several rounds of synthesis are carried out, the double stranded products being denatured between rounds.
  • a thermal stable DNA polymerase is used so that it is not necessary to add enzyme anew for each round of synthesis.
  • the PCR produces a double stranded DNA amplification product which has the same sequence as the original stretch of the met DNA defined by the ends of the primer pair sequences.
  • the amount of PCR product indicates the amount of met DNA or met mRNA in the sample.
  • the product can be detected by a variety of methods well-known in the art.
  • the products are produced in a test tube, or the like, they can be resolved by agarose or polyacrylamide electrophoresis and detected by fluorescence staining, such as ethidium bromide.
  • fluorescence staining such as ethidium bromide.
  • one of the NTPs may be labelled and the PCR products may be determined by measuring incorporation of the labelled NTP.
  • a variety of other methods for resolving, detecting and measuring the amount of PCR product are well-known to the art that are suitable for use in the present invention.
  • In situ techniques may employ the use of fluorescent and radiolabels which can be easily quantitated by fluorescence microscopy or autoradiography, respectively. Generally, fluorescent labels will be preferred.
  • Another labelling technique may employ enzymatic tags which generate readily quantifiable colorimetric or chemiluminescent signals.
  • PCR may be rendered specific for met DNA or met mRNA in in situ and in liquid PCRs.
  • RNAse or DNAse may be used to remove one template or the other from the sample, and the use of primers that distinguish between the gene and the message (e.g. a primer that hybridizes to a sequence in the untranscribed region of the promoter will be gene specific) .
  • primers that distinguish between the gene and the message (e.g. a primer that hybridizes to a sequence in the untranscribed region of the promoter will be gene specific) .
  • PCR is used to detect the presence of met DNA or met mRNA in both normal and tumor tissue. Therefore, when liquid PCR is employed, it is essential to distinguish samples containing normal and tumor tissue. Reduced amounts of met DNA or met mRNA in tumor, relative to normal tissue, is predictive of tumor metastasis.
  • RNA (“Northern") blotting may be used in the methods of the invention.
  • RNA is isolated from tumor and normal breast tissue by any of a number of standard procedures. (Lehrach, H. , Biochemistry, 16: 4743 (1975)) Again, it is important that separate assays be run on the tumor and normal breast tissue so that respective hybridization results can be compared.
  • RNA is subjected to denaturing gel electrophoresis and transferred to nitrocellulose or other support matrix.
  • the met mRNA can be detected by hybridization of radioactively or non- radioactively labelled met .
  • the presence of met mRNA is detected by the intensity of hybridization. The intensity observed in normal tissue is compared with that in tumor tissue; a reduced amount of intensity in tumor tissue is indicative of poor prognosis.
  • the method of the present invention involves the detection of met DNA, met mRNA or Met, the detection and comparison of HGF/SF DNA, mRNA or protein in normal and tumor tissue is likewise predictive of tumor progression, in accordance with the teachings of the present application.
  • Controls were prepared with the C28 antiserum in the presence of competing peptide or without the C28 antiserum. It is known that T47D cells in suspension form lumen ⁇ like structures resembling mammary ducts (I. Keydar et al . , Eur . J . Cancer 15: 659 (1979)). Analyses of T47D cells in paraffin sections stained with C28 antibody showed intense fluorescent staining in cells bordering lumen-like structures, as shown in Figure IA. A marked decrease in intensity was observed when competing C28 peptide was added to the primary antibody or when the primary antibody was omitted.
  • T47D cells stained with the C200 antibody, directed against the met extracellular domain also showed intense fluorescent staining in cells bordering the lumen-like structures, as shown in Figure IA.
  • CLSM a CLSM-like structure
  • Applicants further investigated the subcellular localization of Met by electron microscopy using indirect immunogold labeling and the C28 Met antibody.
  • Met was localized to microvilli that protrude into lumen, as shown in Figure 1C.
  • the intense met-specific staining of cells lining the lumen borders in T47D breast carcinoma cells led the applicants to examine met expression in normal and abnormal human breast tissue.
  • the normal and tumor tissue from a representative breast biopsy is shown in Figure 2.
  • Applicants observed intense Met fluorescence in cells that form a normal mammary duct, as shown in Figure 2A. The intensity of staining was 80-fold greater in the duct-forming cells than in the adjacent nonductal cells.
  • the pattern of Met staining in the mammary duct was similar to, but higher than, that observed in the T47D lumen-like structures. Applicants further discovered that, in contrast, Met staining in adjacent breast tumor tissue was always reduced, but even in the disrupted architecture of the tumor, met fluorescence was evident in lumen- or duct-like structures, as shown in Figure 2C.
  • Met receptor appears to be preferentially expressed in the border cells of the breast duct and the gastrointestinal tract lumen.
  • Met staining in lumen-like structures has been observed in human gastrointestinal lumen and lumen of biliary ducts and esophagus (M. Pratt et al . Int . J . Cancer 49: 323 (1991)).
  • the colocalization of Met staining with anti-P-Tyr immunofluorescence implies that the Met receptor is activated in the lumen-forming cells of the breast duct and gastrointestinal tract, a shown in Figures 2 and 3.
  • HGF/SF human epithelial carcinoma cells in vitro .
  • cells expressing Met were exposed to varying concentrations of HGF/SF (1-100 ng/ml) . More specifically, cells were grown on 16-chamber Labtek slides (Nunc) and treated with specific concentrations of HGF/SF (95% pure, Collaborative Research) for 24 hours. After two washes in PBS, cells were fixed for 10 minutes in cold methanol (-20°C) and washed extensively with PBS. Cells were also stained for 10 minutes with 0.1% methylene blue in PBS and were visualized and photographed using a Zeiss microscope.
  • the grids were washed again in PBS, incubated in RPM11640 medium for 20 min in 1% BSA as above, and reacted with goat anti-rabbit IgG gold (10-nm diameter; 1:10 diluted in 1% BSA; Amersham) at room temperature for 1 hour.
  • the grids were finally washed in PBS and distilled water and stained with uranyl acetate and lead citrate. The sections were observed and photographed using a Philips EM 410 electron microscope.

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Abstract

Procédé de prédiction de la métastase d'une tumeur du sein, selon lequel on détermine la teneur en protéine met des tissus tumoraux par rapport aux tissus normaux des canaux des seins.
PCT/US1993/005952 1992-06-26 1993-06-25 Proto-oncogene met et procede de prediction de l'evolution du cancer du sein WO1994000764A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996005306A2 (fr) * 1994-08-12 1996-02-22 Myriad Genetics, Inc. Mutations et polymorphismes in vivo du gene de la susceptibilite au cancer du sein et des ovaires lie au 17q
EP0699754A1 (fr) * 1994-08-12 1996-03-06 Myriad Genetics, Inc. Méthode de diagnose pour la prédisposition au cancer du sein et des ovaires
US5693473A (en) * 1994-08-12 1997-12-02 Myriad Genetics, Inc. Linked breast and ovarian cancer susceptibility gene
US5709999A (en) * 1994-08-12 1998-01-20 Myriad Genetics Inc. Linked breast and ovarian cancer susceptibility gene
US5753441A (en) * 1994-08-12 1998-05-19 Myriad Genetics, Inc. 170-linked breast and ovarian cancer susceptibility gene
US6162897A (en) * 1994-08-12 2000-12-19 Myriad Genetics, Inc. 17q-linked breast and ovarian cancer susceptibility gene
WO2001007483A1 (fr) * 1999-07-28 2001-02-01 Fbit Ltd. Pronostic et therapie de maladies malignes
US6573043B1 (en) 1998-10-07 2003-06-03 Genentech, Inc. Tissue analysis and kits therefor
US9068011B2 (en) 2010-03-10 2015-06-30 Genmab A+S Monoclonal antibodies against c-Met

Citations (2)

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WO1996005306A2 (fr) * 1994-08-12 1996-02-22 Myriad Genetics, Inc. Mutations et polymorphismes in vivo du gene de la susceptibilite au cancer du sein et des ovaires lie au 17q
EP0699754A1 (fr) * 1994-08-12 1996-03-06 Myriad Genetics, Inc. Méthode de diagnose pour la prédisposition au cancer du sein et des ovaires
WO1996005306A3 (fr) * 1994-08-12 1996-03-14 Myriad Genetics Inc Mutations et polymorphismes in vivo du gene de la susceptibilite au cancer du sein et des ovaires lie au 17q
EP0705903A1 (fr) * 1994-08-12 1996-04-10 Myriad Genetics, Inc. Mutations in vivo et polymorphismes du gène de susceptibilité associé au cancer du sein et des ovaires associé au 17Qq
US5693473A (en) * 1994-08-12 1997-12-02 Myriad Genetics, Inc. Linked breast and ovarian cancer susceptibility gene
US5709999A (en) * 1994-08-12 1998-01-20 Myriad Genetics Inc. Linked breast and ovarian cancer susceptibility gene
US5753441A (en) * 1994-08-12 1998-05-19 Myriad Genetics, Inc. 170-linked breast and ovarian cancer susceptibility gene
US6162897A (en) * 1994-08-12 2000-12-19 Myriad Genetics, Inc. 17q-linked breast and ovarian cancer susceptibility gene
US7344840B2 (en) 1998-10-07 2008-03-18 Genentech, Inc. Tissue analysis and kits therefor
US6573043B1 (en) 1998-10-07 2003-06-03 Genentech, Inc. Tissue analysis and kits therefor
US6905830B2 (en) 1998-10-07 2005-06-14 Genentech, Inc. Tissue analysis and kits therefor
US7129051B2 (en) 1998-10-07 2006-10-31 Genentech Inc Tissue analysis and kits therefor
US7468252B2 (en) 1998-10-07 2008-12-23 Genentech, Inc. Methods for tissue analysis
US7674589B2 (en) 1998-10-07 2010-03-09 Genentech, Inc. Methods for tissue analysis
US7919254B2 (en) 1998-10-07 2011-04-05 Genentech, Inc. Tissue analysis and kits therefor
WO2001007483A1 (fr) * 1999-07-28 2001-02-01 Fbit Ltd. Pronostic et therapie de maladies malignes
US9068011B2 (en) 2010-03-10 2015-06-30 Genmab A+S Monoclonal antibodies against c-Met
US9657107B2 (en) 2010-03-10 2017-05-23 Genmab A/S Monoclonal antibodies against c-Met
US11512140B2 (en) 2010-03-10 2022-11-29 Genmab A/S Monoclonal antibodies against c-Met

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