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WO1996017080A1 - Detection de tumeurs - Google Patents

Detection de tumeurs Download PDF

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Publication number
WO1996017080A1
WO1996017080A1 PCT/GB1995/002734 GB9502734W WO9617080A1 WO 1996017080 A1 WO1996017080 A1 WO 1996017080A1 GB 9502734 W GB9502734 W GB 9502734W WO 9617080 A1 WO9617080 A1 WO 9617080A1
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Prior art keywords
tumour
pcr
cytokeratin
cells
blood
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PCT/GB1995/002734
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English (en)
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Peter John Selby
Susan Ann Burchill
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Imperial Cancer Research Technology Limited
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Publication of WO1996017080A1 publication Critical patent/WO1996017080A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4741—Keratin; Cytokeratin
    • 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
    • G01N33/57484—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates to methods of detecting tumours including metastatic disease in a patient, particularly metastatic disease of epithelial cell tumour origin, more particularly disseminating colon carcinoma.
  • malignant tumours or cancers commonly results in the release of some of the cancerous cells from the developing tumour into the blood or other body fluids. These cells are then transported to other parts of the body where they may become implanted and set up secondary tumours or metastases, thus leading to a general dissemination or spread of the original cancer that is responsible for production of the primary tumour.
  • metastasis may commence at quite an early stage in the development and growth of the primary tumour, and in fact it is metastases, frequently haematogenous metastases produced by cancer tumour cells circulating in the peripheral blood, that determine the outcome of the disease for most patients.
  • GB 2 260 811 A discloses a general method for the diagnosis or monitoring of cancer of a malignant tumour using a RT-PCR.
  • melanoma cells were detected in blood of a patient using a RT-PCR.
  • Specific oligonucleotide primers were used to amplify tyrosinase mRNA.
  • Tyrosinase is not expressed in normal blood but is expressed at a relatively high level in some melanoma cells (which are derived from melanocytes); however, analysis of tyrosinase gene expression is complicated by the presence of tyrosinase-related protein genes and the method is limited because some melanomas lack tyrosinase expression (amelanotic tumours).
  • Burchill et al (1994) Int. J. Cancer SI, 671-675 describes neuroblastoma cell detection by RT-PCR for tyrosine hydroxylase mRNA.
  • melanoma, neuroblastoma and CML are tumours arising from highly specialised cell types and, in the case of CML, arising from a cell containing chromosomal abnormality.
  • Some of the most prevalent cancers arise from less specialised epithelial cells, for example breast cancer and colon carcinoma.
  • any method of detecting cells in the blood derived from such epithelial cell tumours must rely on the marker to be detected not being expressed in normal blood.
  • Cytokeratins are components of mammalian cell cytoskeleton and constitute a multigene family of proteins (see Nagel (1988) Am. J. Surg. Pathol. 12, (suppl. 1), 4-16) and Moll et al (1982) Cell 31, 11-24 for reviews). CKs are expressed predominantly in epithelial cells where they show strict lineage and differentiation-associated patterns of expression. Malignant cells generally retain the CKs of their progenitor cell type and have been used previously to characterise neoplastic cells of epithelial origin.
  • CK8 and CK18 are expressed in all tissue that has been studied including peripheral blood mononuclear cells, aspirated bone marrow cells and lymph nodes. Some of these cells are components of normal blood and so CK8 and CK18 are expressed in normal blood and, consequently, are of no use in analysing blood samples for the presence of cells derived from an epithelial cell tumour.
  • CK19 like CK8 and CK18, is not a suitable target for detection of tumour cells in peripheral blood.
  • CK20 is not expressed in normal blood samples.
  • CK20 is expressed, for example, in a high proportion of colorectal carcinomas, stomach cancers, mucinous ovarian adenocarcinomas and transitional cell carcinomas.
  • One aspect of the present invention provides a method of determining whether a human patient has a tumour or whether a tumour has metastasised comprising the steps of (1) obtaining a sample of tissue from the patient, the said tissue being one that does not normally contain a cytokeratin 20 (CK20) gene product and (2) determining whether a cytokeratin 20 (CK20) gene product is present in said tissue sample.
  • CK20 cytokeratin 20
  • determining whether a tumour has metastasised we include determining whether any tumour cells are found at a site remote from a primary tumour whether or not those tumour cells are found in a further tumour.
  • the method includes determining the likelihood that a tumour has spread or is spreading or will spread in a patient.
  • the invention comprises determining whether a tissue sample from a patient contains a cytokeratin 20 (CK20) gene product, particularly a tissue sample which does not normally contain a cytokeratin 20 (CK20) gene product.
  • CK20 gene product we include CK20 mRNA and CK20 protein.
  • CK20 gene is polymorphic and therefore mat many allelic forms occur.
  • CK20 gene we include all allelic forms. Different allelic forms can be readily detected by comparing one CK20 gene or mRNA sequence with another, for example by DNA sequencing or restriction fragment length polymorphism (RFLP) analysis.
  • RFLP restriction fragment length polymorphism
  • tissue sample is blood or bone marrow.
  • Blood is readily obtained from the patient using venepuncture whereas bone marrow is obtained using standard aspiration techniques (for example by placing a needle into the iliac bone and aspirating). Purged bone marrow is suitable.
  • lymph nodes, peripheral stem cell harvests, urine and cystectomy samples may be used.
  • urine in the term "tissue sample” .
  • a suitable sample also includes polymorphonuclear cells such as neutrophils.
  • the amount of CK20 gene product present in the tissue (such as blood) of a human patient who is determined by the method of the invention to have CK20 gene product present in said tissue, compared to a human who does not have a CK20 gene product (as defined), is at least two-fold higher, preferably at least 10-fold higher, more preferably at least 100-fold higher and most preferably at least 1000-fold higher.
  • tumour is an epithelial cell tumour.
  • epithelial cell-derived tumours including breast carcinoma colorectal carcinoma, stomach adenocarcinoma, mucinous ovarian adenocarcinoma, all bladder carcinoma including dysplasia and transitional cell carcinoma.
  • the vast majority of mucinous ovarian and colorectal adenocarcinomas express CK20; greater than 75% of stomach and gall bladder adenocarcinomas contain CK20-expressing cells; and more than half of pancreatic adenocarcinomas contain CK20-expressing cells.
  • tumour is a lung tumour.
  • a high proportion of Merkel cell carcinomas and transitional cell carcinomas express CK20.
  • the method of the invention is particularly suited to determine whether a patient has, or is likely to develop metastatic disease from, mucinous ovarian and colorectal adenocarcinomas.
  • the patient has not suffered a local trauma or has not undergone surgery both of which may result in the release of epithelial cells into the blood.
  • Presence of a cytokeratin 20 gene product can be determined in a tissue sample using various techniques. Conveniently, it is determined by detecting CK20 messenger RNA (mRNA).
  • CK20 mRNA can be detected in a tissue sample by hybridising a specific nucleic acid probe to the mRNA (such as an antisense RNA or antisense oligonucleotide, the said probe being labelled with a readily-detectable moiety for example, a fluorescent dye or a radionuclide), the sensitivity of such a method may not allow the detection of the very small amount of CK20 mRNA that is present in a tissue sample.
  • a 5 ml blood sample may contain only tens of CK20-expressing cells derived from the epithelial cell tumour but tens of thousands of cells in total.
  • the mRNA is detected by, first, making a complementary DNA (cDNA) copy of the CK20 mRNA and, second, amplifying the cDNA using DNA amplification methods.
  • cDNA complementary DNA
  • a tissue sample is obtained from the patient. Conveniently, 2 ml of blood is removed from the patient and frozen at -80°C.
  • Total cellular RNA is extracted from the blood sample, for example using standard techniques described in Sambrook et al (1989)
  • UltraspecTM RNA (a trade mark) available from Biogenesis, Bournemouth, UK.
  • the mRNA is converted into cDNA using suitable oligonucleotide primers, deoxynucleotides and an enzyme with reverse transcriptase activity.
  • suitable oligonucleotide primers deoxynucleotides and an enzyme with reverse transcriptase activity.
  • the oligonucleotide for making cDNA can be, for example, either:
  • oligo-dT primer which hybridises to the polyA tail of mRNA
  • Random short sequences such as random hexamers, which prime cDNA synthesis of the total RNA
  • Oligonucleotides specific for CK20 which prime cDNA synthesis from CK20 mRNA.
  • any residual chromosomal DNA is removed using a nuclease such as DNase I.
  • Oligonucleotide primers are selected which direct specific amplification of the CK20 cDNA. A PCR is performed using these oligonucleotides, a thermal-stable DNA polymerase and deoxynucleotides. 6. Optionally, further oligonucleotides are selected which direct specific amplification of a DNA fragment chosen from the inter- primer region defined in step 5 and a further PCR (a "nested" PCR) is carried out.
  • step 7 The product of the PCR reaction of step 5 or 6 is detected using agarose gel electrophoresis and ethidium bromide staining of the DNA.
  • Nucleotide sequences of CK20 cDNAs are shown in Figures 5 to 7.
  • the cDNA and gene sequence described in Moll et al (1993) Differentiation 53, 75-93 is incorporated herein by reference as are the sequences and information from the relevant database submissions described in the legends to Figures 5 to 7.
  • the approximate position of the introns (in the gene) are marked.
  • oligonucleotides suitable for DNA amplification comprise a sequence selected from the sequence shown in any one of Figures 5 to 7 such that the first oligonucleotide is capable of hybridizing to one exon and the second oligonucleotide is capable of hybridizing to another exon.
  • the oligonucleotide primers are between 10 and 50 nucleotides in length, more preferably between 14 and 30 nucleotide long, most preferably around 20 nucleotides long.
  • the oligonucleotide primers can hybridize to all alleles of the CK20 gene. Regions of the CK20 gene and cDNAs common to all alleles can be determined by comparing the sequences of CK20 genes and cDNAs such as those shown in Figures 5 to 7.
  • Preferred oligonucleotide primers comprise or consist of the sequence 5'-
  • CTCCTGGAATCTCCAATGG SEQ ID No 3
  • GCATTTTGCAGTTGAGCATCC SEQ ID No4
  • TGGGCAACACTGTCAATGTGG SEQ ID No 11
  • TCCATGTTACTCCGAATCTGC SEQ ID No 12
  • PCR primers do not contain any complementary structures with each other longer than 2 bases, especially at their 3' ends, as this feature may promote the formation of an artifactual product called "primer dimer”.
  • primer dimer When the 3' ends of the two primers hybridize, they form a “primed template” complex, and primer extension results in a short duplex product called “primer dimer”.
  • Optimum annealing temperatures may be determined empirically and may be higher than predicted.
  • Tag DNA polymerase does have activity in the 37-55°C region, so primer extension will occur during the annealing step and the hybrid will be stabilized.
  • concentrations of the primers are equal in conventional (symmetric) PCR and, typically, within 0.1- to 1- ⁇ M range.
  • a labelled oligonucleotide capable of hybridising to the amplified DNA as a probe.
  • the oligonucleotide probe hybridises to the interprimer sequence as defined by the two primers.
  • the oligonucleotide probe is preferably between 10 and 50 nucleotides long, more preferably between 15 and 30 nucleotides long.
  • the probe may be labelled with a radionuclide such as 32 P, 33 P and 35 S using standard techniques, or may be labelled with a fluorescent dye.
  • the amplified DNA product may be detected in solution (see for example Balaguer et al (1991) "Quantification of DNA sequences obtained by polymerase chain reaction using a bioluminescence adsorbent" Anal. Biochem. 195, 105-110 and Dilesare et al (1993) "A high-sensitivity electrochemiluminescence- based detection system for automated PCR product quantitation” BioTechniques 15, 152-157.
  • Any of the DNA amplification protocols can be used in the method of the invention including the polymerase chain reaction, QB replicase and ligase chain reaction.
  • the polymerase chain reaction is particularly preferred because of its simplicity.
  • the polymerase chain reaction may, if desired, be carried out in situ, or at least in whole tissue samples isolated from the patient, using the methods described in VMwareinoth et al (1994) Pathol. Res. Pract. 190(1), 1017-1025, incorporated herein by reference; and
  • CK20 gene product In principle it is possible to detect the presence of a CK20 gene product by determining whether the sample contains any CK20 protein. This is most conveniently achieved using antibodies that react specifically with CK20.
  • monoclonal antibody K 5 20.8 reactive against CK20 can be purchased from Cymbus Bioscience Limited, Southampton, UK or other suitable monoclonal antibodies can be made using methods well known in the art.
  • suitable monoclonal antibodies to CK20 may be prepared using the techniques described in Monoclonal Antibodies: a manual of techniques, H. Zola (CRC Press, 1988) and in Monoclonal Hybridoma Antibodies: Techniques and Applications, J.G.R. Hurrell (CRC Press, 1982).
  • the antibody is labelled with a readily detectable marker such as a radionuclide or fluorescent dye.
  • a radionuclide or fluorescent dye such as "Tc, ,23 I and 32 P.
  • Suitable fluorescent dyes include fluorescein.
  • a human patient has a tumour or is likely to develop metastatic disease from such a tumour
  • the physician can decide on an appropriate course of treatment.
  • Other methods may supplement the present method in order to reach a diagnosis.
  • a further aspect of the invention provides a kit of parts comprising oligonucleotide primers capable of amplifying a cytokeratin 20 (CK20) cDNA, deoxynucleotides and a DNA polymerase.
  • oligonucleotide primers capable of amplifying a cytokeratin 20 (CK20) cDNA, deoxynucleotides and a DNA polymerase.
  • the kit may further comprise means for extracting RNA suitable primers for making CK20 cDNA, an enzyme with reverse transcriptase activity, and means for detecting an amplified DNA product.
  • Figure 1 shows the products of RT-PCR for CK8, 19 and 20 mRNA on 1 pg to 100 ng of total mRNA isolated from RT112 (CK8), MCF7 (CK19) and HT29 (CK20) cell lines.
  • a single band of 244, 214 and 370 bp respectively was identified after separation of products in an agarose gel and staining with ethidium bromide. There was an increase in the intensity of this band with increasing RNA concentration.
  • M molecular weight markers
  • W water control
  • Figure 2 shows the products of RT-PCR for CK8(i), 19(ii) and 20(iii) mRNA separated by agarose gel electrophoresis and stained with ethidium bromide in 6 control bloods (1-6).
  • FIG. 3 shows the products of RT-PCR for CK20 mRNA separated by electrophoresis and stained with ethidium bromide in 6 control bone marrow samples (i). RT-PCR for GAP-DH in the same RNA samples (ii).
  • C positive control of RNA extracted from HT29 cells.
  • W water negative control.
  • M molecular weight markers.
  • Figure 4 shows the products of RT-PCR for CK20 mRNA separated by agarose gel electrophoresis and stained with ethidium bromide in blood samples spiked with 1 to 10 s HT29 cells. A single 370 bp band was identified when as few as 100 cells per ml of whole blood were analysed
  • +C positive control of RNA extracted from HT29 cells.
  • W water negative control.
  • M molecular weight markers.
  • Figure 5 shows the nucleotide sequence of part of a human CK20 mRNA with the amino acid sequence of the encoded polypeptide composed of the exon sequence taken from Accession X73501, (bases 1 to 18061).
  • Figure 6 shows the nucleotide sequence of part of a human CK20 mRNA with the amino acid sequence of the encoded polypeptide composed of the exon sequence taken from as in
  • Figure 7 shows the nucleotide sequence of part of a human CK20 mRNA taken from Accession X73502
  • Reference: 1 bases 1 to 1461
  • the three well-characterised human cell lines used in the study were the transitional cell carcinoma-derived RT112 cell line, the breast adenocarcinoma MCF-7 cell line and the colonic adenocarcinoma HT29 cell line.
  • MCF-7 and HT29 cells express CK8, CK18 and CK19 (Moll et al (1982) Cell 31, 11-24), whereas RT112 express CK8, CK18, CK19 along with other CK isotypes characteristic of bladder epithelial cells (Wu et al (1982) Cell 31, 693-703).
  • CK20 the most recently identified CK isotype, is expressed by HT29 cells, but not by MCF-7 cells (Moll et al (1992) Am. J. Pathol. 140, 427-447). All cell lines were maintained in a 1:1 mixture of DMEM and RPMI 1640 media supplemented with 5 % foetal bovine serum and passaged using 0.25% trypsin in versene (0.02% EDTA).
  • Blood and bone marrow samples Normal blood or bone marrow samples were obtained from volunteers aged between 18 to 45 years. Samples were taken into EDTA, aliquoted into 2 ml volumes and frozen at -80°C until required for RNA extraction.
  • RNA extraction Total cellular RNA was extracted from cell lines, normal whole blood, normal bone marrow or spiked normal blood using UltraspecTM RNA (Biogenesis, Bournemouth, UK) according to the manufacturer's instructions.
  • RT-PCR Reverse transcriptase-polymerase chain reaction
  • Reverse transcriptase negative controls in which reverse transcriptase enzyme was omitted were included for all RT-PCR reactions.
  • Water negative controls contained all components for the RT-PCR reaction but no targeted RNA.
  • positive controls (+C) of RNA extracted from HT29, MCF7 or RT112 cells were included.
  • Molecular weight markers ⁇ X 174 RF DNA/Hae III, Gibco BRL, Paisley, Scotland or 123 bp ladder, Pharmacia, Milton Keynes, UK) were included on all agarose gels.
  • Primer sequences for PCR were selected using the Dieffenbach Selection Programme. Primers were located within different exons and were either 20, 21 or 22 mers. Specificity of RT-PCR. RT-PCR products were separated on agarose gels and Southern blotted onto nylon membrane (Hybond N + , Amersham, UK). Filters were hybridised with a gamma 32 P end-labelled oligonucleotide probe, the sequence of which lay between each primer set. The nucleotide sequence of RT-PCR products was confirmed by dideoxy chain termination sequencing (Sequenase, USS, Canada).
  • RNA (100 pg) from HT29 cells was included as a positive control.
  • RT-PCR detection of bladder, breast and colon carcinoma cells RT-PCR for CK8 generated a single 244 bp band identified on ethidium bromide stained agarose gels (Fig l ,i). This fragment was confirmed by Southern blot analysis and sequencing (data not shown) to be the fragment of CK8 which lies between the two primers used for PCR. The band was detected in 10 pg-100 ng of total RNA from RT112 cells.
  • RT-PCR for CK19 generated a single 214 bp band identified on ethidium bromide stained agarose gels (Fig 1 ,ii) which was confirmed to be CK19 by Southern blot analysis and sequencing (data not shown). This band was detected in 100 pg-1 ng of total RNA from MCF7 cells.
  • RT-PCR for CK20 generated a single band of 370 bp (Fig l,iii). This band was confirmed by Southern hybridisation and sequence analysis (data not shown) and detected in 100 pg of total RNA from HT29 cells.
  • RNA In all three cases there was an increase in band intensity with increasing amounts of RNA (Fig 1). No transcripts were identified in water control samples (Fig 1) or RT negative samples (results not shown).
  • CK8 and CK19 RT-PCR products were identified under the described conditions. Southern blotting confirmed amplified bands were CK8 and CK19 RT-PCR products.
  • CK20 was undetectable by ethidium bromide staining or Southern blot hybridisation.
  • RT-PCR results for CK8(i), CK19(ii) or CK20(iii) are shown in Fig 2 for six control bloods, 6/6 were positive for CK8, 3/6 for CK19 and 0/6 for CK20.
  • RT-PCR for CK20 in 6/6 normal bone marrow samples showed no amplified bands (Fig 3,i). The integrity of bone marrow RNA samples was confirmed by RT-PCR for GAP-DH (Fig 3,ii).
  • HT29 cell spiking In HT29 cell spiking experiments it was possible to detect down to 100 HT29 cells diluted in 2 ml of whole human blood (Fig 4,i). The 370 bp band generated was shown by Southern blotting to hybridise to a 3 P end-labelled oligonucleotide probe specific for CK20 and confirmed by sequence analysis to be that of CK20 (results not shown). No RT-PCR products were detected in whole blood alone (Fig 4,i 0). RT-PCR products were not identified in reverse transcriptase negative samples (Fig 4,ii).
  • CK20 mRNA was not detected in any normal blood or bone marrow samples examined suggesting it to be the CK of choice for detection of carcinomas of epithelial origin.
  • CK20 has been detected in almost all cases of colorectal adenocarcinomas by immunohistochemistry and is a useful target for the detection of disseminating colon carcinoma by RT- PCR.
  • Example 2 Determining whether a patient has an epithelial cell tumour
  • RNA is produced from the blood as described in Example 1 and a RT-PCR is carried out using the CK20-specific primers as described in Example 1. If a CK20-specific DNA product is amplified it suggests that the patient may have an epithelial cell tumour. Further confirmatory tests may be performed in order to reach a diagnosis.
  • Example 3 Determining whether a patient with colorectal carcinoma tumours is likely to develop metastases
  • RNA is produced from the blood as described in Example 1 and a RT-PCR is carried out using the CK20-specific primers as described in Example 1. If a CK20-specific DNA product is amplified it suggests that the patient is likely to develop metastatic disease disseminated from the colorectal tumour. Further confirmatory tests may be performed in order to reach a diagnosis.

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Abstract

Un procédé pour déterminer si un patient est atteint d'une tumeur ou si la tumeur s'est métastasée consiste (1) à prélever un échantillon de tissu du patient qui ne contient normalement pas un produit du gène de la cytokératine 20 (CK 20), et à (2) déterminer si un produit du gène de la cytokératine 20 (CK 20) est présent dans l'échantillon de tissu. Lorsqu'on a déterminé si le patient a une tumeur ou si sa tumeur est susceptible de se métastaser, le médecin peut décider de la thérapie qu'il convient d'appliquer.
PCT/GB1995/002734 1994-11-26 1995-11-24 Detection de tumeurs WO1996017080A1 (fr)

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GB9423912A GB9423912D0 (en) 1994-11-26 1994-11-26 Detecting tumours
GB9423912.6 1994-11-26

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Cited By (14)

* Cited by examiner, † Cited by third party
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WO1998020166A3 (fr) * 1996-11-06 1998-10-22 Sequenom Inc Diagnostics de l'adn fondes sur la spectrometrie de masse
DE19716346C1 (de) * 1997-04-18 1998-11-19 Christoph Prof Dr Wagener Verfahren zum Nachweis von Cytokeratinen
WO1999010528A1 (fr) * 1997-08-22 1999-03-04 Michael Giesing Procede permettant de caracteriser des cellules cancereuses disseminees et micrometastasees
WO2000055628A1 (fr) * 1999-03-12 2000-09-21 Oxford Glycosciences (Uk) Ltd. Proteines permettant le diagnostic et le traitement du cancer du sein
US6258538B1 (en) 1995-03-17 2001-07-10 Sequenom, Inc. DNA diagnostics based on mass spectrometry
EP0972830A4 (fr) * 1997-02-21 2003-02-05 Takara Shuzo Co Genes associes au cancer
WO2003097878A1 (fr) 2002-05-21 2003-11-27 Sysmex Corporation Amorces d'amplification d'acide nucleique destinees a detecter des cytokeratines et technique d'examen utilisant ces amorces
WO2004023110A1 (fr) * 2002-08-14 2004-03-18 Hexal Gentech Forschungsgmbh Procede de detection immunocytologique ou moleculaire de cellules tumorales disseminees dans un liquide corporel et kit associe
EP0960214A4 (fr) * 1996-12-06 2004-08-04 Urocor Inc DIAGNOSTIC D'ETAT PATHOLOGIQUE A L'AIDE DE PROFILS d'ARN MESSAGER
US6949633B1 (en) 1995-05-22 2005-09-27 Sequenom, Inc. Primers useful for sizing nucleic acids
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WO2007020081A1 (fr) * 2005-08-17 2007-02-22 Medexis S.A. Composition et methode pour la determination de l'expression de ck-19
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US7501251B2 (en) 1996-11-06 2009-03-10 Sequenom, Inc. DNA diagnostics based on mass spectrometry
WO1998020166A3 (fr) * 1996-11-06 1998-10-22 Sequenom Inc Diagnostics de l'adn fondes sur la spectrometrie de masse
EP0960214A4 (fr) * 1996-12-06 2004-08-04 Urocor Inc DIAGNOSTIC D'ETAT PATHOLOGIQUE A L'AIDE DE PROFILS d'ARN MESSAGER
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WO1999010528A1 (fr) * 1997-08-22 1999-03-04 Michael Giesing Procede permettant de caracteriser des cellules cancereuses disseminees et micrometastasees
WO2000055628A1 (fr) * 1999-03-12 2000-09-21 Oxford Glycosciences (Uk) Ltd. Proteines permettant le diagnostic et le traitement du cancer du sein
EP1510587A4 (fr) * 2002-05-21 2007-11-14 Sysmex Corp Amorces d'amplification d'acide nucleique destinees a detecter des cytokeratines et technique d'examen utilisant ces amorces
WO2003097878A1 (fr) 2002-05-21 2003-11-27 Sysmex Corporation Amorces d'amplification d'acide nucleique destinees a detecter des cytokeratines et technique d'examen utilisant ces amorces
US8076080B2 (en) 2002-05-21 2011-12-13 Sysmex Corporation Nucleic acid amplification primers for detecting cytokeratins and examination method with the use of the primers
WO2004023110A1 (fr) * 2002-08-14 2004-03-18 Hexal Gentech Forschungsgmbh Procede de detection immunocytologique ou moleculaire de cellules tumorales disseminees dans un liquide corporel et kit associe
US7323304B2 (en) * 2002-08-14 2008-01-29 Hexal Gentech Forschungsgmbh Method for the immunocytological or molecular detection of disseminated tumor cells from a body fluid and kit that is suitable therefor
WO2007020081A1 (fr) * 2005-08-17 2007-02-22 Medexis S.A. Composition et methode pour la determination de l'expression de ck-19
US9593374B2 (en) 2005-08-17 2017-03-14 Lianidou Evriklia Composition and method for determination of CK19 expression
EP1845167A1 (fr) * 2006-04-11 2007-10-17 Roche Diagnostics GmbH Procédé de détermination rapide de cytokératine 19 (CK19) et les amorces et les sondes correspondantes

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