EP1200623A2 - Methode diagnostique - Google Patents

Methode diagnostique

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Publication number
EP1200623A2
EP1200623A2 EP20000946122 EP00946122A EP1200623A2 EP 1200623 A2 EP1200623 A2 EP 1200623A2 EP 20000946122 EP20000946122 EP 20000946122 EP 00946122 A EP00946122 A EP 00946122A EP 1200623 A2 EP1200623 A2 EP 1200623A2
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EP
European Patent Office
Prior art keywords
nucleotide sequence
wtl
subject
specific
tumour
Prior art date
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EP20000946122
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German (de)
English (en)
Inventor
Karim Malik
Keith Brown
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University of Bristol
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University of Bristol
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Publication date
Priority claimed from GBGB9916669.6A external-priority patent/GB9916669D0/en
Priority claimed from GBGB9926293.3A external-priority patent/GB9926293D0/en
Application filed by University of Bristol filed Critical University of Bristol
Publication of EP1200623A2 publication Critical patent/EP1200623A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12Q2600/154—Methylation markers

Definitions

  • This invention relates to a diagnostic method, to a nucleotide sequence comprising a Wilms' tumour suppressor gene (WTl) antisense regulatory region, and to a method of disease detection and prognosis based on the methylation state of the regulatory region.
  • WTl Wilms' tumour suppressor gene
  • WT Wilms' tumour
  • the human WTl gene resides on chromosome l lpl3 (Call et al, (1990) Cell 60, p 509-520; Gessler et al, (1990) Nature 343, p774-778; Call et al, (1994), US5,350,840) and is genomically organised as 10 exons spanning a 60 kilobase chromosomal region. Intragenic deletions and mutations of the tumour suppressor gene, WTl, have been detected in approximately 10% of Wilms' tumours.
  • WTl gene expression is controlled in a highly specific manner, increasing as metanephric mesenchymal cells progress towards immature epithelial cells, and attenuating as the cells become more phenotypically mature.
  • the inverse correlation between WTl expression and the differentiation status of human leukaemic cells along with evidence of expression in ovary and testis and the spinal chord and brain strongly suggest that the function of the WTl gene product may be pivotal in growth and/or differentiation in a variety of cell types.
  • the WTl protein which includes four zinc fingers, is expressed as four isoforms arising from two alternative splice sites (I and ⁇ ) in the gene.
  • Splice II occurs within the zinc finger domain, inserting or omitting three amino-acids (KTS) between zinc fingers 3 and 4.
  • KTS three amino-acids
  • WTl -KTS specifically binds to the EGR site consensus sequence (5'-GCGGGGGCG-3') whereas the WTl protein with KTS (WTl+KTS) does not.
  • EGR early growth response gene
  • IGF-II insulin-like growth factor type II
  • PDGF-A platelet derived growth factor A
  • CSF-1 colony stimulating factor- 1
  • EGF-R epidermal growth factor receptor
  • the human WTl promoter region has been characterised and found to belong to the family of TATA-less, CCAAT-less, GC-rich promoters with multiple responsive sites for the transcription factor Spl. EGR/WTl consensus sequences were also identified upstream and downstream of the major transcriptional start site (Hofmann et al, (1993) Oncogene 8, 3123-3132) and the suggestion that these sites may allow WTl autorepression was subsequently verified using transient transfection assays with the human promoter (Malik et al, (1994) FEBS Letters 349, 75-78)
  • WTl function is crucial in the normal development of the urogenital system, as demonstrated in WAGR (Wilms tumour, Aniridia, Genitourinary abnormalities and mental Retardation) syndrome and in Denys-Drash syndrome (DDS), diseases characterised by renal and genital abnormalities together with a predisposition to Wilms' tumour (reviewed in Coppes et al, (1993) FASEB J. 7, 886-895.)
  • antisense WTl mRNA transcripts with no apparent open reading frames have been detected in foetal kidney and WTs, suggesting a regulatory role for these mRNAs (Campbell et al, (1994) Oncogene 9, 583-595; Eccles et al, (1994) Oncogene 9, 2059-2063).
  • One such function of these mRNAs may be the formation of RNA heteroduplexes with sense WTl mRNA, thereby modulating the finite levels of cellular WTl protein.
  • the inventors reported the identification of an antisense WTl promoter located in intron 1 which is activated by WTl.
  • the WTl antisense transcript may upregulate the levels of WTl protein (Moorwood et al, (1998) J. Pathol 185, 352-359), and aberrations of the control mechanisms for antisense RNA transcription may result in inappropriate temporal and spatial expression of WTl protein, in turn contributing to tumourigenesis.
  • WTl can increase the tumour growth rate of adenovirus-transformed baby rat kidney cells (Menke et al, (1996) Oncogene 12, 537-546).
  • WTl antisense promoter locus was identified as a hypermethylated sequence in human breast cancers (Huang et al, (1996) Cancer Res. 57, 1030-1034) and breast cancers have been shown to have decreased expression of WTl (Silberstein et al, (1997) Proc. Natl Acad. Sci. USA 94, 8132-8137).
  • the inventors have identified an antisense regulatory region (ARR) of the WTl antisense promoter, and have demonstrated that the ARR is part of a differentially methylated region.
  • the WTl ARR characterised and utilized as the basis of the invention is structurally and functionally distinct from previously described WTl gene sequences (for example Call et al, (1994), US patent 5,350,840).
  • the inventors have found a correlation between the levels of ARR methylation, and the pathological state of human cells, Specifically, a variety of cancer cells are shown to differ from their normal counterparts on the basis of epigenetic changes.
  • a first aspect of the invention provides a nucleotide sequence encoding a WTl antisense regulatory region comprising at least a portion of, or consisting of, the sequence shown in SEQ1, or at least a portion of a variant, due to base substitutions, deletions and/or additions, of the sequence shown in SEQ.l.
  • a second aspect of the invention provides a nucleotide sequence encoding a WTl antisense regulatory region comprising or consisting of the sequence shown in SEQ2, or at least a portion of a variant, due to base substitutions, deletions and/or additions, of the sequence shown in SEQ.2.
  • the WTl antisense regulatory region may be limited to the portion of sequence shown in bold in SEQ. 2, or variants of such a sequence due to base substitutions, deletions and/or additions.
  • a third aspect of the invention provides a nucleotide sequence encoding a WTl antisense regulatory region negative regulatory element (NRE) comprising at least a portion of the sequence shown in SEQ.l or at least a portion of a variant, due to base substitutions, deletions, and/or additions, of the sequence shown in SEQ.l .
  • the nucleotide sequence shown in SEQ.1 may contain several WTl antisense regulatory region negative regulatory elements.
  • a nucleotide sequence according to the first, second or third aspects of the invention is a DNA or RNA sequence. Portions of any sequences are preferably functional i.e. they have a biological function of a corresponding native sequence.
  • a fourth aspect of the invention provides a method of disease detection, diagnosis or prognosis in a subject with cancer, using the differentially methylated state of specific nucleotide sequences, such as the nucleotide sequences in the WTl ARR region.
  • Genomic epigenetic changes are often regional, for example affecting a variety of gene loci on chromosome l lpl5 (Feinberg (1999) Cancer Research (suppl.) 59, p 1743-1746).
  • the inventors' identification of the chromosome 1 lpl3 region as a target for epigenetic changes by methylation therefore suggest that other DNA probes/DNA sequences from the l ip 13 region, including those derived from the l ip 13 genes reticulocalbin and PAX6, may also be utilized for detection purposes in methods according to the invention.
  • the specific nucleotide sequence(s) may be one or more regulatory elements preferably one or more negative regulatory elements (NRE), for example, one or more NREs within the ARR.
  • the NRE sequence or sequences may be part of the WTl gene, or part of the chromosome l lpl3 region, such that a method of disease diagnosis and prognosis in a subject diagnosed with cancer, comprises determining the methylation state of a NRE, or an ARR, of the WTl gene or chromosome 1 lpl3 region DNA sequence in the subject, and correlating the methylation state of the NRE with the diagnosis and expected long-term recovery prognosis of the subject.
  • hypermethylation of the NRE indicates that the subject has a positive long term recovery prognosis
  • hypomethylation of the NRE indicates that the subject is predisposed to relapsing after treatment
  • hypermethylation of the NRE indicates that the subject has a positive long term recovery prognosis
  • hypomethylation of the NRE indicates that the subject is predisposed to relapsing after treatment.
  • hypomethylation is detected specifically in tumours, and in colorectal cancer cell lines, hypomethylation correlates with tumouri genie potential.
  • hypermethylation of the specific nucleotide sequence or sequences may indicate the presence of cancer cells and/or a predisposition of the subject to relapsing after treatment
  • hypomethylation of the specific nucleotide sequence or sequences may indicate the absence of cancer cells and/or that the subject has a positive long term recovery prognosis
  • the diagnostic application is underlined by the hypomethylation in WTs, as opposed to the hypermethylation of other renal tumours, such as primitive neuroectodermal tumour (PNET) and clear cell sarcoma of the kidney (CCSK) (see figure ID).
  • PNET primitive neuroectodermal tumour
  • CCSK clear cell sarcoma of the kidney
  • the methylation state may be determined by restriction of the WTl antisense regulatory region using enzymes such as ifoA 12361, Spel and Kpnl in combination.
  • Bshl236I is an isoschizomer of Bst UI. Bshl236I cuts at the restriction sequence CGCG only when there is no CpG methylation. Methylated sequences are not restricted by 2?.sA 12361. Therefore, the restriction pattern obtained for a nucleotide sequence which has been restricted with Bshl236I gives a different band pattern depending on whether the Bshl236I sites in the nucleotide sequence are methylated or not. Other commercially available enzymes may also be used, with one or more being able to distinguish between methylated and unmethylated DNA.
  • the methylation state may be determined using a PCR-based assay system.
  • a PCR-based assay system may involve the use of sodium-metabisulphite. This has the effect of converting all unmethylated cytosine residues to uracil residues.
  • the PCR reaction uses the following primers to amplify at least a portion of the WTl antisense regulatory region:
  • Tf 5 '-GGGTGGAGAAGAAGGATATATTTAT-3 ' .
  • Tr 5'-TAAATATCAAATTAATTTCTCATCC-3'.
  • TfN 5'-GATATATTTATTTATTAGTTTTGGT-3' (nested primer).
  • TrN 5'-AAACCCCTATAATTTACCCTCTTC-3' (nested primer).
  • the conditions used in the PCR reaction are the same as the conditions mentioned later in the specification.
  • the PCR products obtained from the PCR reaction, as described below, may then be cloned and sequenced.
  • the PCR products may be cloned into a vector such as pGEM-T (Promega).
  • the PCR products may be sequenced directly. Once sequenced, any methylated cytosine residues will remain readable as 'C in the nucleotide sequence, whereas unmethylated cytosines will appear as T residues in the sequence.
  • the nested PCR reaction involves the following primers.
  • Tf 5 ' -GGGTGGAGAAG AAGG AT ATATTT AT-3 ' .
  • Tr 5'-TAAATATCAAATTAATTTCTCATCC-3'.
  • a fifth aspect of the invention provides a method of cancer detection in cells derived from a subject comprising detection of tumour-specific alteration of genomic imprinting. Any bi-allelic expression of tumour-specific genes may indicate the presence of tumourgenic cell proliferation if the normal tissue expresses the gene monoallelically. Alternatively, with some cancers, the normal tissue may be biallelic, and the cancer monoallelic. Additionally, methylation changes may be accompanied by changes in gene expression through silencing or enhanced gene expression, irrespective of allelic contributions to gene dosage (reviewed in Jones (1996), Cancer Research 56, p2463-2467)
  • tumour-specific alteration of genomic imprinting may be detected by reverse transcription PCR (RT-PCR).
  • RT-PCR reverse transcription PCR
  • the method may be used in the detection of WT in a subject, and may detect alteration of genomic imprinting of WT-specific genes such as the WT-1 gene.
  • the altered genomic imprinting detected may be relaxation of genomic imprinting, loss of imprinting, or gain of imprinting.
  • the RT-PCR may use two primers designed to anneal to a tumour-specific gene sequence on opposite sides of an allelic polymophism which introduces a restriction-site in one allele only.
  • the RT-PCR may use the following primers:
  • Primer 1 WTl 8 [CTTAGCACTTTCTTCTTGGC]
  • Primer 2 WITKBF2 [TTGCTCAGTGATTGACCAGG]
  • a sixth aspect of the invention provides a method of treating a subject with a specific cancer, comprising altering the genomic imprinting of a tumour-specific gene. This may involve relaxation of the genomic imprinting, or reversal of relaxed genomic imprinting.
  • a seventh aspect of the invention provides a diagnostic kit, assay or monitoring method using a method according to a fifth aspect of the invention.
  • An eighth aspect of the invention provides a method of detection of the methylation state of a WTl antisense regulatory region comprising detection of a tumour-specific alteration in genomic imprinting using a method according to a preceding aspect of the invention, and correlating a detected alteration in genomic imprinting with differential methylation of the WTl antisense regulatory region. For example, relaxation of genomic imprinting may be correlated with hypomethylation of the WTl antisense regulatory region.
  • Figure 1(A) shows the probe used for the detection of methylation for Southern blotting
  • Figure 1(B) shows a Southern blot of three acute myelogenous leukaemia (AML) DNAs and a normal peripheral blood lymphocyte DNA; and
  • Figure 1(C) shows a Southern blot of DNAs from a non-tumourogenic and a highly- tumourgenic colorectal cell line
  • Figure 1(D) shows a Southern blot of matched normal kidney and WT samples, matched normal kidney and PNET or CCSK DNAs and a foetal kidney control
  • Figure 1(E) shows Southern blot analysis of breast tumour DNAs for changes in the ARR methylation status.
  • Figure 2 shows the nucleotide sequence of a WTl ARR, with the primer hybridisation sites indicated by arrows;
  • Figure 3(A) is a schematic diagram showing the primers on either side of the antisense
  • Figure 3(B) shows a southern blot of the antisense WTl RNA RT-PCR products
  • SEQ.l shows a nucleotide sequence of the WTl ARR
  • SEQ.2 shows a nucleotide sequence of a negative regulatory element of a gene encoding
  • SEQ.3 shows the nucleotide sequence of a WTl antisense region (Gessler, M & Bruns
  • the WTl cDNA and WTl promoter region were cloned from a human foetal kidney cDNA library (Clontech) and a human B-cell genomic library ( ⁇ Sha2001, kindly supplied by T. H. Rabbitts, Medical Research Council, Cambridge) respectively.
  • Plaque screen filters Du Pont
  • Genomic clones corresponding to the 5 '-end of the WTl gene were subcloned and characterised by restriction analysis according to standard methodology (Sambrook, J., et al (1989). Molecular Cloning, Vols 1 and 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.). DNA sequences were determined by the dideoxy chain terminator method (Sanger, F., et al (1977). Proc. Natl Acad. Sci. USA, 74, 5463-5467) and by ⁇ taq cycle-sequencing according to the manufacturers instructions (USB- Amersham).
  • Figure 1(D) shows a Southern blot of matched normal kidney and Wilms' tumour samples. All WT samples were confirmed as having no loss of heterozygosity. Also shown are matched normal kidney and PNET or CCSK DNAs.
  • hypomethylation of specific nucleotide sequences correlates with the tumour state. However, in other cancers, this correlation may be inverted, such that hypermethylation of specific nucleotide sequences corresponds to the methylation status of tumour cells, and hypomethylation may indicate normal cells.
  • FIG 1(E) An example of this is shown in figure 1(E) , with Southern blot analysis of normal breast tissue DNA and breast tumour DNAs for changes in the ARR methylation status.
  • Four infiltrating ductal carcinomas of varying aggressiveness all showed increased methylation of the WTl ARR compared to the normal breast tumour DNA. Therefore the relative differential methylation comparing normal tissue and tumour tissue may be utilised diagnostically.
  • Tumour cells and normal cells may be distinguished by their epigenotype as previously outlined.
  • Knowledge of the DNA sequence of the WTl antisense regulatory region has made it possible to develop a PCR-based assay system to allow the determination of the methylation status of samples, which will require less biological material.
  • This method involves introducing CpG dinucleotides which are not part of a restriction enzyme recognition sequence by treatment of genomic DNA samples with sodium-metabisulphite (Merck), thereby converting all unmethylated cytosine residues to uracil (Merck), thereby converting all unmethylated cytosine residues to uracil (Merck), thereby converting all unmethylated cytosine residues to uracil (Paulin, R., et al (1998) Nucleic Acids Research 8, 4777-4790). Specific regions of interest in the WTl intronic sequence can then be amplified using primers specific for both strands of DNA.
  • PCR bands obtained can be directly sequenced or cloned using a commercially available vector such as pGEM-T (Promega) and analysed by DNA sequencing. Any methylated cytosine residues will remain readable as 'C in the DNA sequence, whereas unmethylated cytosines will appear as 'T'.
  • nested primers which include one specific for the methylated Bshl236I site shown to be commonly differentially methylated (boxed in Figure 2), or one specific for the unmethylated Bsh 12361 site (i.e. specific for C— » conversion) may be employed, permitting discrimination between methylated and non-methylated sequences by visualisation of the PCR products, i.e. if a primer specific for the methylated Bsh 12361 site is used, a PCR product will only be observed if the Bsh 12361 site in the sample is methylated, otherwise, no PCR amplification will occur.
  • Illustrative primers which may be used for methylation-specific PCR are shown below, and their hybridisation positions to the WTl sequence are shown by arrows in Figure 2 for top-strand amplification. Allowing for C— »T conversion, these are:
  • Tf 5'-GGGTGGAGAAGAAGGATATATTTAT-3 '.
  • TfN 5'-GATATATTTATTTATTAGTTTTGGT-3' (nested primer).
  • TrN 5'-AAACCCCTATAATTTACCCTCTTC-3' (nested primer).
  • Typical primary amplifications are conducted with Amplitaq (Perkin-Elmer) with 100 ng. of bisulphite-treated DNA in buffer supplemented with 3mM MgCI 2 .
  • Amplification conditions are 3 mins. denaturation at 94°C, followed by 35 cycles of denaturation at 94°C for 30 sees, annealing at 50°C for 30 sees, and extension at 72°C for 90 sees. A final extension of 5 mins at 72°C completes the reaction.
  • Secondary PCR with the nested primers employs the same conditions, but using l/lOO" 1 of the primary PCR reaction and 24 cycles.
  • the inventors have detected a correlation between the methylation state of the ARR and the diagnosis and long term disease prognosis in subjects with cancer.
  • the diagnostic potential is shown by the hypomethylation in WTs, as opposed to the hypermethylation of other renal tumours, such as primitive neuroectodermal tumour (PNET) and clear cell sarcoma of the kidney (CCSK) (see figure ID).
  • PNET primitive neuroectodermal tumour
  • CCSK clear cell sarcoma of the kidney
  • the methylation state of the NRE can be used as a potential early indicator of the long term diseased prognosis.
  • Subjects who have an unmethylated NRE can be kept under closer observation for early detection of relapse. This will maximise their chances for recovery.
  • the expense of such close observation post-treatment is not necessary with subjects with unmethylated NRE, as these patients are expected to respond well to treatment once any relapse has been detected by normal routine checking.
  • hypomethylation of specific nucleotide sequences corresponds to a predicted positive long term prognosis of the subject with the AML
  • hypomethylation corresponds to a predisposition of the subject to relapsing after treatment.
  • this correlation may be inverted, such that hypermethylation of specific nucleotide sequences corresponds with a predisposition to relapsing after treatment, and hypomethylation may indicate a positive long term prognosis for recovery. Therefore, decisions on the best methods of therapy to suit the subject can be made in the light of an educated expectation of how the subject is expected to respond to treatment in the event of a relapse of their cancer condition.
  • WTl ARR/NRE Antisense Regulatory Region/Negative Regulatory Region
  • Genomic imprinting is the phenomenon by which maternal or paternal copies of a gene can be selectively expressed, with methylation of DNA serving as the regulatory signal. Loss of such a signal can lead to an altered dosage of gene expression that can be deleterious to normal cell growth.
  • the IGF2 gene exhibits loss of genomic imprinting control of IGF2 and is overexpressed in WTs (Feinberg, A. P. (1999) Cancer Res. (suppl), 59: 1743s- 1746s). As IGF2 is a growth factor, this may easily contribute to uncontrolled proliferation associated with tumourigenesis.
  • RT-PCR reverse transcription-PCR
  • Typical reaction conditions used for the RT-PCR were annealing of the reverse primer to 1 ⁇ g of total RNA by heating to 60°C for 5 mins, followed by quenching on ice, followed by reverse transcription carried out with Super RT (HT Biotechnologies, Cambridge, U.K.) reverse transcriptase at 50°C for 60 mins. This was followed by PCR cycling as follows:
  • PCR products obtained were digested by adding the restriction enzyme Mnll directly to the PCR mix and incubating for 60 minutes at 37°C.
  • the PCR products were then separated on 2% agarose gels and then alkali blotted onto Hybond N + membrane and hybridised with a 32 P-labelled antisense cDNA probe.
  • the sequence of the probe is shown in bold between WTl 8 and WITKBP2 in SEQ. 3.
  • the following primers were used as DNA controls:
  • Primer 1 WITKBF2 [TTGCTCAGTGATTGACCAGG]
  • Primer 2 WITKBR2 [TTGGCTGGAAAGCTTGCAGC]
  • the Mnll polymorphism (Grubb, G. R. et al (1995) Oncogene, 10: 1677-1681) utilised is marked by an asterisk in figure 3 A, and results in RT-PCR products of 286 and 222bp for biallelic expression, or alternatively major allelic bands of 286bp or 222bp for monoallelic expression.

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Abstract

La présente invention concerne une méthode permettant de détecter un cancer chez un individu par la détection d'une modification de son empreinte génomique, ainsi qu'une méthode permettant de déterminer le pronostic à long terme d'un sujet chez lequel on a diagnostiqué un cancer, sur la base des changements des états de méthylation d'une séquence nucléotidique spécifique.
EP20000946122 1999-07-15 2000-07-17 Methode diagnostique Withdrawn EP1200623A2 (fr)

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GBGB9916669.6A GB9916669D0 (en) 1999-07-15 1999-07-15 Diagnostic method
GB9916669 1999-07-15
GB9926293 1999-11-05
GBGB9926293.3A GB9926293D0 (en) 1999-11-05 1999-11-05 Diagnostic method
PCT/GB2000/002741 WO2001006005A2 (fr) 1999-07-15 2000-07-17 Methode diagnostique

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EP1352089B1 (fr) * 2000-08-25 2006-12-06 Lovelace Respiratory Research Institute Methode de detection de cancer par reaction de polymerisation en chaine specifique de la methylation imbriquee
JP2005514956A (ja) * 2002-01-18 2005-05-26 ジェンザイム・コーポレーション 胎児ｄｎａの検出および対立遺伝子の定量化のための方法
GB0209776D0 (en) * 2002-04-29 2002-06-05 Univ Bristol Diagnostic method
FR2854903A1 (fr) * 2003-05-13 2004-11-19 Centre Nat Rech Scient Utilisation des loci bage (b melanoma antigens) comme marqueurs tumoraux
EP3194624B1 (fr) 2014-09-15 2022-02-16 Garvan Institute of Medical Research Méthodes pour le diagnostic, le pronostic et la surveillance du cancer du sein et réactifs correspondants

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WO1991007509A1 (fr) * 1989-11-13 1991-05-30 Massachusetts Institute Of Technology Localisation et caracterisation du gene tumoral de wilms
AU8381698A (en) * 1997-07-03 1999-01-25 Case Western Reserve University Compositions and methods for detection of genomic imprinting disorders

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