WO1999020168A2 - Mutations de lignee germinale dans le gene de la e-cadherine et procede de detection d'une predisposition au cancer - Google Patents

Mutations de lignee germinale dans le gene de la e-cadherine et procede de detection d'une predisposition au cancer Download PDF

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WO1999020168A2
WO1999020168A2 PCT/NZ1998/000160 NZ9800160W WO9920168A2 WO 1999020168 A2 WO1999020168 A2 WO 1999020168A2 NZ 9800160 W NZ9800160 W NZ 9800160W WO 9920168 A2 WO9920168 A2 WO 9920168A2
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cadherin
cancer
alteration
predisposition
gene
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PCT/NZ1998/000160
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WO1999020168A3 (fr
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Anthony Edmund Reeve
Parry John Guilford
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University Of Otago
Te Whetu Whanau Trust Limited
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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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to methods by which a predisposition to cancer can be determined.
  • it relates to methods for detecting whether a patient has a predisposition to cancer, particularly hereditary diffuse gastric cancer.
  • the key to cancer treatment is early detection.
  • the ability to predict who is at extreme risk, before the appearance of clinical symptoms, will enable the earliest possible detection of malignancy (watchful waiting). It will also enable prophylactic intervention prior to the onset of clinical signs.
  • Gastric cancer remains a major cause of cancer death worldwide, and about 10% of cases show familial clustering. The relative contributions of inherited susceptibility and environmental effects to familial gastric cancer are poorly understood because little is known of the genetic events that predispose to gastric cancer.
  • the invention broadly provides a method of testing to detect whether an individual is predisposed to cancer which comprises the step of detecting the presence or absence of an alteration (mutation) in the gene encoding E-cadherin.
  • the invention provides a method of assessing the risk in a human subject for a predisposition for cancer which comprises the step of determining whether there is a germline alteration in the gene encoding E-cadherin, wherein the presence of an alteration is indicative of a risk for a predisposition for cancer.
  • gene encoding E-cadherin means not only the coding sequence for wild-type E-cadherin but also includes non-coding flanking sequences and regulatory elements, mutations in which can cause transcript instability and/ or transcriptional repression, and the sites for transcript splicing. These include the two nucleotides immediately upstream (usually "AG”) and the two nucleotides immediately downstream (usually "GT”) of each exon, and also the splicing branch site located 18-38 bp upstream of each exon.
  • AG the two nucleotides immediately upstream
  • GT two nucleotides immediately downstream
  • the presence or absence of the mutation is detected through analysis of the DNA encoding E-cadherin and/or its regulatory elements.
  • the presence or absence of the mutation is detected through analysis of mRNA transcribed from the DNA encoding E-cadherin.
  • the presence or absence of the mutation is detected through analysis of the amino acid sequence of the expressed E-cadherin protein.
  • the invention provides a method of prophylaxis and/ or therapeutic treatment against cancer of an individual identified as having a risk of predisposition to cancer by a method defined above which comprises the step of increasing, maintaining and/ or restoring the active concentration of wild- type E- cadherin protein within said individual.
  • the method will be a gene therapy method and will involve supplying the individual with wild-type E-cadherin gene function.
  • Figure 1 shows the nucleotide and amino acid sequences for wild-type E- cadherin cDNA
  • Figure 2 is a kindred map for one family (family A) having a predisposition to gastric cancer. Numbers to the right of the symbols indicate age at death. The age is underlined if a blood or biopsy sample was available. General symbols: squares, males; circles, females; all symbols with a diagonal, deceased. Solid symbols: gastric carcinoma, pathology available; dotted symbols: gastric carcinoma, pathology unavailable; vertical stripes: colorectal cancer;
  • Figure 3 is a graph showing the age of death from gastric cancer in the studied kindred of family A
  • Figure 4 shows the results of a mutation analysis of exon 7 of the E- cadherin gene as follows:
  • Figure 5 is an abbreviated kindred map for a second family (family B).
  • General symbols squares, males; circles, females; all symbols with a diagonal, deceased.
  • Solid symbols gastric carcinoma, pathology available; dotted symbols: gastric carcinoma, pathology unavailable; vertical stripes: colorectal cancer. Diagonal hatching: unconfirmed gastric carcinoma;
  • Figure 6 shows sequence analysis results for DNA from family B ( Figure 6 A) and family C ( Figure 6B) , exons 15 and 13 respectively;
  • Figure 7 shows pedigrees of non-Maori gastric cancer families. General symbols: squares, males; circles, females; all symbols with a diagonal, deceased. Patient numbers are included; and
  • Figure 8 shows mutations in gastric cancer families, (a). Exon 11 DNA sequence from family 1000 showing the insertion of an additional C nucleotide between the G at position 1588 and the A at position 1591. (b). Exon 2 sequence from family 4201 showing the heterozygous (G/T) mutation at position 70. (c). Exon 8 / intron 8 sequence of family CHG 72 showing the heterozygous (G/A) mutation at the first nucleotide of the intron. Nucleotide positions are as described in Berx et al. (1995). Sequencing products were analysed on a LiCor 4000L DNA sequencer.
  • the method of the invention detects a predisposition to cancer.
  • the critical finding made by the applicants is that this predisposition is due to an alteration (mutation) in the gene encoding E-cadherin. This finding forms the basis of the present invention.
  • E-cadherin is a transmembrane protein with five tandemly repeated extracellular domains and a cytoplasmic domain which connects to the actin cytoskeleton via a complex with ⁇ , ⁇ and ⁇ catenins (Grunwald (1993)). It plays an important role in establishing cell polarity and maintaining normal tissue morphology and cellular differentiation. Diminished E-cadherin expression is associated with poorly differentiated carcinomas which display aggressive histopathologic characteristics such as infiltrative growth and lymph node involvement (Shiozaki et al. (1995)). Under- expression has been proposed as a prognostic marker of poor clinical outcome in many tumour types (Bracke et al. (1996)). In experimental tumour models, restored expression of E-cadherin can suppress the invasiveness of epithelial tumour cells (Frixen (1991), Vlemincke (1991)).
  • the gene encoding E-cadherin was identified as a susceptibility gene through genetic linkage analysis. This analysis was performed in relation to samples obtained from a large (Maori) kindred from New Zealand, the pedigree pattern of which is shown in Figure 2 (family A). This pedigree pattern is consistent with the dominant inheritance of a susceptibility gene with incomplete penetrance.
  • the linkage analysis determined that the susceptibility to cancer was associated with the gene encoding E-cadherin. This was confirmed with reference firstly to two further Maori kindreds (families B and C) and then to non-Maori kindreds.
  • alteration of the wild-type E- cadherin gene is detected.
  • the method can be performed by detecting the wild-type E-cadherin gene and confirming the lack of a predisposition or neoplasia.
  • “Alteration of a wild-type E-cadherin gene” encompasses all forms of mutations including deletions, insertions and point mutations in the coding and noncoding regions. Deletions may be of the entire gene or only a portion of the gene. Point mutations may result in stop codons, frame shift mutations or amino acid substitutions.
  • the alterations or mutations which are focus of the predictive method of the invention are germline mutations. Germline mutations can be found in any of a body's tissues and are inherited.
  • Point mutation events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the E-cadherin gene product, or a decrease in mRNA stability or translation efficiency.
  • Predisposition to cancers can be ascertained by testing any tissue of a human for mutations of the E-cadherin gene. For example, a person who has inherited a germline E- cadherin mutation would be prone to develop cancers. This can be determined by testing DNA from any sample from the person's body such as serum, sputum and urine. Most simply, blood can be drawn and DNA extracted from the cells of the blood. In addition, prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic fluid for mutations of the E-cadherin gene.
  • a preliminary analysis to detect deletions in DNA sequences can be performed by looking at a series of Southern blots of DNA cut with one or more restriction enzymes, preferably a large number of restriction enzymes. Each blot contains DNA from a series of normal individuals and from a series of test cases. Southern blots displaying hybridizing fragments (differing in length from control DNA when probed with sequences near or including the E-cadherin locus) indicate a possible mutation. If restriction enzymes which produce very large restriction fragments are used, then pulsed field gel electrophoresis ("PFGE”) can be employed.
  • PFGE pulsed field gel electrophoresis
  • Detection of point mutations may be accomplished by molecular cloning of the E- cadherin allele(s) and sequencing that allele(s) using techniques well known in the art.
  • the gene sequences can be amplified, using known polynucleotide amplification techniques, directly from a genomic DNA preparation from the sample tissue.
  • the amplification techniques which can be used include methods such as the polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. These methods are well known and widely practised in the art.
  • E-cadherin sequences generated by amplification may be sequenced directly.
  • the amplified sequence (s) may be cloned prior to sequence analysis.
  • a method for the direct cloning and sequence analysis of enzymatically amplified genomic segments has been described by Scharf, 1986.
  • SSCA single stranded confirmation analysis
  • DGGE denaturing gradient gel electrophoresis
  • RNase protection assays Finkelstein et al, 1990; Kinsler et al, 1991
  • ASO's allele-specific oligonucleotides
  • allele-specific PCR primers are used which hybridize at their 3' ends to a particular E-cadherin mutation. If the particular E-cadherin mutation is not present, an amplification product is not observed.
  • Amplification Refractory Mutation System As disclosed in European Patent Application Publication No. 0332435 and in Newton et al, 1989. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification.
  • RFLP restriction fragment length polymorphism
  • RFLP restriction fragment length polymorphism
  • SSCA detects a band which migrates differentially because the sequence change causes a difference in single- strand, intramolecular base pairing.
  • RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments.
  • DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel.
  • an allele-specific oligonucleotide assay an oligonucleotide is designed which detects a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal.
  • the protein beings only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.
  • Mismatches are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of samples.
  • RNase protection method An example of a mismatch cleavage technique is the RNase protection method. This method involves the use of a labeled riboprobe which is complementary to the human wild- type E-cadherin gene coding sequence. The riboprobe and either mRNA or DNA isolated from the test tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch.
  • RNA product when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA.
  • the riboprobe need not be the full length of the E-cadherin mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the E-cadherin mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.
  • DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, eg., Cotton et al, 1989; Shenk et al, 1975; Novack et al,, 1986.
  • mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See eg. Cariello, 1988.
  • riboprobes or DNA probes the cellular mRNA or DNA which might contain a mutation can be amplified using PCR before hybridization. Changes in DNA of the E-cadherin gene can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.
  • DNA sequences of the E-cadherin gene which have been amplified by use of PCR may also be screened using allele-specific probes.
  • These probes are nucleic acid oligomers, each of which contains a region of the E-cadherin gene sequence harboring a known mutation.
  • one oligomer may be about 30 nucleotides in length, corresponding to a portion of the E-cadherin gene sequence.
  • Hybridization of allele-specific probes with amplified E-cadherin sequences can be performed, for example, on a nylon filter such as Hybond. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same mutation in the tumour tissue as in the allele-specific probe.
  • Mutations from potentially susceptible patients falling outside the coding region of E-cadherin can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the E-cadherin gene.
  • An early indication that mutations in noncoding regions are important may come from Northern blot experiments that reveal messenger RNA molecules of abnormal size or abundance in cancer patients as compared to control individuals.
  • E-cadherin mRNA expression can be detected by any techniques known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild- type E-cadherin gene. Alteration of wild-type E-cadherin genes can also be detected by screening for alteration of wild- type E-cadherin protein. For example, monoclonal antibodies immunoreactive with wild-type E-cadherin can be used to screen a tissue with lack of bound antigen indicating an E-cadherin mutation.
  • Monoclonal antibodies with affinities of 10- 8 M- 1 or preferably 10- 9 to 10- 10 M- 1 or stronger will typically be made by standard procedures as described, eg. in Harlow & Lane, 1988 or Goding, 1986. Briefly, appropriate animals will be selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalised myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supematants of each clone tested for then- production of an appropriate antibody specific for the desired region of the antigen.
  • recombinant immunoglobulins may be produced using procedures known in the art (see, for example, US Patent 4,816,567).
  • the antibodies may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in the literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149; and 4,366,241.
  • Antibodies specific for products of mutant alleles could also be used to detect mutant E-cadherin gene product. Such antibodies can be produced in equivalent fashion to the antibodies for wild-type E-cadherin as described above.
  • the immunological assay in which the antibodies are employed can involve any convenient format known in the art. Such formats include Western blots, immunohistochemical assays and ELISA assays. In addition, functional assays such as protein binding determinations, can also be used.
  • any approach to detecting a germline alteration in the underlying DNA coding for wild-type E-cadherin expression can be employed, whether the analysis be of the DNA itself, mRNA transcribed from the DNA or the protein which is the ultimate expression product of the DNA.
  • Genotyping DNA extracted from blood and biopsy samples (Banerjee et al, (1995)) was genotyped using standard conditions (Dib, (1996)) in reactions containing 0.2U Ampl ⁇ Taq Gold (Perkin Elmer) and 25 pmole of infrared labelled (IR41) forward primer (MWG Biotech). Products were analysed on a LiCor 4000L DNA sequencer.
  • SSCP analysis was carried out as described by Berx et al, (1995). The PCR products were electrophoresed at room temperature through a 6% non denaturing polyacrylamide gel without added glycerol. Products were detected by autoradiography.
  • Nucleotide position 1008 was PCR- amplified from the cDNA using a forward primer within exon 7 (5'-TAA CAG GAA CAC AGG AGT CAT CA-3') and a reverse primer from exon 8 (5'-GTG GTG GGA TTG AAG ATC GG-3').
  • Plasmid and direct sequencing RT-PCR products were eluted from a 6% polyacrylamide denaturing gel, re-amplified with the original primers using Pwo polymerase (Boehringer Mannheim) and ligated into the i ⁇ coRV site of Bluescript. Template for direct sequencing of mutations was produced from genomic DNA by PCR using the SSCP antisense primers and the sense primers 8 (Berx et al, 1995) with an added 5' leader corresponding to the T3 sequencing primer. Plasmid and direct sequencing were carried out using Thermosequenase (Amersham) and an IR41 labelled (MWG Biotech) T3 primer (3 pmoles/reaction). The products were analysed on a LiCor 4000L DNA sequencer.
  • Lod scores were calculated assuming either equal allele frequencies or, in a conservative approach (in the absence of allele frequencies for the study population), using the actual frequencies observed in the study kindred.
  • GDB Human Genome Database, Baltimore (Maryland, USA): John Hopkins University. Dib, 1996.
  • exon-linking RT-PCR (exons 7-8) was performed on stomach biopsy material taken from an affected family member. In addition to the expected product of 180 bp, a minor 187 bp band was also observed. Both products were cloned and resulting clones sequenced. 10/ 10 clones derived from the larger band contained the mutation and a 7 bp insertion of intronic DNA. The insertion is a consequence of splicing at a cryptic splice site (Oda et al, (1994)).
  • transcript which is incorrectly spliced at exon 7 is unstable in vivo, the extent of aberrant splicing was estimated from the proportion of correctly spliced transcript which contained the G to T mutation. 1/ 14 clones derived from the 180 bp product contained the mutation. This result demonstrates that, relative to the wild- type transcript, only about 15% of the mutant transcript accumulates in stomach tissue.
  • Glu 336 is located in one of the LDRE motifs which form part of E-cadherin's four calcium binding pockets. Calcium binding is required for dimerisation and rigidification of E-cadherin and provides protection from proteolytic degradation (Nagar et al, (1996)). Molecular modelling indicates that an Asp at position 336 would cause a significant deformation in the calcium binding pocket with a probable negative effect on its ability to bind calcium (data not shown). The fact that the LDRE motif is conserved, not only amongst vertebrates but also in Drosophila (Mahoney et al, (1991)), suggests that a Glu to Asp mutation at this position is not tolerated.
  • the proband of family C (aged 30 years) showed an SSCP band in exon 13.
  • Direct sequencing identified a heterozygous C ⁇ T transition at nucleotide 2,095 which converted Gin 699 to a TAG stop codon (Fig. 6B). This inactivating mutation would result in an expressed E-cadherin peptide lacking both the transmembrane and cytoplasmic domain.
  • Family 1000 is of mixed Northern European ancestry (Fig. 7a).
  • the proband and her mother were both diagnosed with high grade adenocarcinoma with signet ring histology and linitis plastica at ages 40 and 48, respectively.
  • the proband's maternal grandfather had died of cancer of unknown type at age 45.
  • a maternal aunt was diagnosed at age 59 with a scirrhous adenocarcinoma of the left breast.
  • she also had resection of an adenocarcinoma of the cardia of the stomach. Microscopic examination of the gastric tumour showed a diffuse, poorly differentiated mucous producing adenocarcinoma with numerous signet ring cells.
  • Fig. 7b Family 4201 (Fig. 7b) is of European origin. The family has a strong history of gastric and breast cancer and leukemia. Pathology specimens were available from three of four individuals affected by gastric cancer (III- l, 111-2, III- 5). These three cancers were all diffusely infiltrative signet ring adenocarcinomas (Watanabe et al, (1990)). Extensive thickening of the stomach wall, consistent with linitis plastica, was described in one case (III- l). The age at diagnosis of gastric cancer in this family ranged from 37 to 46 years and the age at death ranged from 39 to 55 years. One obligate carrier is unaffected by cancer at age 71 years.
  • her sister (II-2) was diagnosed with gastric cancer at age 37 and breast cancer two years later. Two cases of breast cancer alone, and one of Kaposi's sarcoma in the brain (associated with HIV infection) have occurred in this family, with ages at diagnosis of 39, 46 and 40 years, respectively. The histology of these tumours was unavailable. In addition, three family members had unspecified leukemia diagnosed at ages 66, 45, and 45 years. A fourth case of leukemia occurred in a spouse at age 83 years.
  • Family CHG 72 is of African American origin and has had four family members affected by gastric cancer.
  • the age of diagnosis of the cancers was 25 to 58 with the patients dying between ages 29 and 58.
  • the tumours were all diffuse, poorly differentiated infiltrative adenocarcinomas with signet ring histology.
  • a half sister (II- 1) to the proband died of an unconfirmed cancer in her thirties and a child (IV- 1) currently aged 10 years suffers from aplastic anemia.
  • DNA manipulation DNA was extracted from blood using either standard techniques or the Puregene kit (Gentra Systems, Minneapolis, Minnesota) following the manufacturer's protocol. DNA extractions from paraffin-embedded, formalin-fixed tissue were carried out using previously reported techniques (Greer et al., (1995); Grady et al, (1998)). All tumours from family 4201 and family CHG 72 were microdissected prior to DNA extraction. PCR products for the 16 E-cadherin exons were amplified using 1U AmpliTaq Gold (Perkin Elmer) and the primers and conditions described by Berx et al. (1996). A 5' leader corresponding to the T3 sequencing primer was added to the sense primer.
  • primers 5'-TTC CCC CAC CCC AGG TCT C-3' (EX2F) and 5'-CCC TCA CCT CTG CCC AGG AC-3' (EX2R), correspond to nucleotides 1- 19 and 136- 117 of the exon 2 genomic sequence (accession # L34937), respectively. Sequencing was performed using either EX2F or the primer 5'-TGT AGC TCT CGG CGT CAA AG-3' (complementary to nucleotides 93- 1 12 of the E-cadherin cDNA sequence (Berx et al, 1995)). The sequencing products were electrophoresed on a 6% polyacrylamide 7M urea gel at 70W (50°C) for 50-90 minutes and visualized using either autoradiography or a Storm 820 Phosphorimager (Molecular Dynamics).
  • Sequencing genomic DNA from peripheral white blood cells of the proband of family 4201 identified a heterozygous G->T transversion at nucleotide 70 (70G->T) in exon 2 (Fig. 8b).
  • the proband is unaffected but is an obligate carrier of the predisposing mutation.
  • the mutation would convert a glutamic acid (Glu24) to a TAG stop codon in the signal peptide of the E-cadherin precursor protein.
  • This mutation was also identified in microdissected normal tissue from gastric biopsies of three siblings (III- l, III-2, III-5) with gastric cancer, and peripheral white blood cell DNA from an unaffected sibling (III-4) and a first cousin (III-7) affected by breast cancer. DNA from blood of one unaffected family sibling (III-6) showed no mutation. No other biological samples were available from any of the other family members.
  • the E-cadherin gene was PCR amplified using peripheral white blood cell DNA from the proband of family CHG 72 (II-4). Sequencing identified a heterozygous G->A transition in the splice donor site of intron 8 ( 1 137+ 1G->A) . Guanine at the + 1 position of the splice consensus sequence is 100% conserved in eukaryotic splice sites (Padgett et al, ( 1986)). The G->A change would be predicted to result in either skipping of exon 8 or the activation of cryptic splice sites. This mutation was identified in DNA from normal and microdissected tumour tissue from paraffin blocks in three additional affected family members (II-2, II-3, III- l).
  • LOH heterozygosity
  • the present invention will therefore mean that people from families with histories of familial cancer (such as HDGC) will be able to undergo tests which will search for the presence of E-cadherin gene mutations.
  • familial cancer such as HDGC
  • E-cadherin as a cancer susceptibility gene has implications beyond early detection.
  • E- cadherin Compounds which can increase the expression, or prevent the decrease, of E- cadherin would be potential cancer chemopreventative agents for carriers of mutations in this gene.
  • a number of chemicals are already known to up-regulate E- cadherin:
  • the gene portion should encode a part of the E-cadherin protein which is required for non- neoplastic growth of the cell. More usual is the situation where the wild-type E- cadherin gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant E-cadherin gene present in the cell. Such recombination requires a double recombination event which results in the correction of the E-cadherin gene mutation.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used.
  • the wild-type E-cadherin gene or fragment may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cancer cells.
  • gene therapy is particularly appropriate for use in pre-cancerous cells, in which the level of E-cadherin polypeptide is absent or diminished compared to normal cells. It may also be useful to increase the level of expression of a given E-cadherin gene even in those cells in which the mutant gene is expressed at a "normal" level, but the gene product is not fully functional.
  • Gene therapy would be carried out according to generally accepted methods, for example as described by Kren et al, (1998), or as described by Friedman in Therapy or Genetic Disease, T. Friedman, ed., Oxford University Press (1991), pp 105- 121.
  • Cells from a patient would be first analyzed by the methods described above, to ascertain the production of E-cadherin polypeptide.
  • a virus or plasmid vector, containing a copy of the E-cadherin gene linked to expression control elements and capable of replicating inside the target cells, is prepared. Suitable vectors are known, such as disclosed in US Patent 5,252,479 and PCT published application WO 93/07282.
  • the vector is then injected into the patient, either locally at the site of the target cells or systemically (in order to reach any target cells that may be at remote sites). If the transfected gene is not permanently incorporated into the genome of each of the targeted cells, the treatment may have to be repeated periodically.
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods. These include viral and nonviral transfer methods.
  • viruses have been used as gene transfer vectors, including papovaviruses (eg. SV40, Madzak et al., (1992)), adenovirus (Berkner (1992)), vaccinia virus (Moss (1992)), adeno-associated virus (Muzyczka (1992)), herpesviruses including HSV and EBV (Margolskee (1992); Johnson et al, (1992); Fink et al, (1992); Breakfield and Geller, (1987); Freese et al, (1990)), and retroviruses of avian (Petropoulos et al, (1992), murine (Miller (1992)); and human origin (Shimada et al, (1991); Helseth et al, (1990); Page et al, (1990); Buchschacher and Pangani
  • Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Pellicer et al, (1980)); mechanical techniques, for example microinjection (Anderson et al, (1980)); membrane fusion-mediated transfer via liposomes (Lim et al, (1992)); and direct DNA uptake and receptor- mediated DNA transfer (Wolff et al, (1990); Wu et al, (1991)).
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the target cells.
  • the retroviral vector producer cell line can be injected into the patient (Culver et al, 1992). Injection of producer cells would then provide a continuous source of vector particles.
  • plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector.
  • the trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged.
  • Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is nonspecific, localized in vivo uptake and expression have been reported in tumour deposits, for example, following direct in situ administration (Nabel, 1992).
  • Peptides which have wild- type E-cadherin activity can be supplied to cells which carry mutant or missing E- cadherin alleles as an alternative approach to gene therapy.
  • Such peptides can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors and known techniques (Sam brook et al, (1989)).
  • E-cadherin polypeptide can be extracted from E-cadherin-producing mammalian cells.
  • the techniques of synthetic chemistry can be employed to synthesize E-cadherin protein (Merryfield, (1963)).
  • Active E-cadherin molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Extracellular application of the E-cadherin gene product may be sufficient to prevent tumour growth. Supply of molecules with E- cadherin activity should lead to partial reversal of the risk of a later neoplastic state. Other molecules with E-cadherin activity (for example, peptides, drugs or organic compounds) may also be used to effect such a reversal. Modified polypeptides having substantially similar function can also be used for peptide therapy.
  • cells which carry a mutant E-cadherin allele can be used as model systems to study and test for substances which have potential as prophylactic /therapeutic agents.
  • the cells are typically cultured epithelial cells. These may be isolated from individuals with E-cadherin mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the E-cadherin allele.
  • the neoplastically transformed phenotype of the cell is determined. Any trait of neoplastically transformed cells can be assessed, including anchorage- independent growth, tumourigenicity in nude mice, invasiveness of cells, and growth factor dependence. Assays for each of these traits are known in the art.
  • Vleminckx K., Vakaet, L., Mareel, M., Fiers, W. & Roy, F. V. Genetic manipulation of E-cadherin expression by epithelial tumour cells reveals an invasion suppressor role. Cell 66, 107- 119 (1991).
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO. 1: gcttgcggaa gtcagttcag actccagccc gctccagccc ggcccgaccc gaccgcaccc 60 ggcgcctgccc ctcgctcggc gtcccggcc agccatgggc ccttggagcc gcagcctctc 120 ggcgctgctgctgctgctgctgc aggtctcctctgc caggagccgg agccctgcca 180 ccctggcttt gacgccgaga gctacacgtt cacggtgcccccacc tggagagagg 240 c
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO. 4: Wggg at tgaagatcgg
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO. 5: ttccccacc ccaggtctc
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO. 6: ccctcacctc tgcccaggac (2) INFORMATION FOR SEQ ID NO. 7:
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO. 7: tgtagctctc ggcgtcaaag

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  • General Engineering & Computer Science (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

L'invention concerne des procédés de détermination d'une prédisposition au cancer. Elle concerne notamment des procédés permettant de détecter si un patient présente une prédisposition à un cancer, notamment le cancer gastrique diffus héréditaire, par référence à une altération (mutation) se produisant dans le gène codant la E-cadhérine.
PCT/NZ1998/000160 1997-10-17 1998-10-19 Mutations de lignee germinale dans le gene de la e-cadherine et procede de detection d'une predisposition au cancer WO1999020168A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU97679/98A AU9767998A (en) 1997-10-17 1998-10-19 Germline mutations in the e-cadherin gene and method for detecting predispos ition to cancer

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NZ328994 1997-10-17
NZ32899497 1997-10-17

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WO1999020168A2 true WO1999020168A2 (fr) 1999-04-29
WO1999020168A3 WO1999020168A3 (fr) 1999-07-01

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001002860A1 (fr) * 1999-07-01 2001-01-11 Consejo Superior De Investigaciones Cientificas Snail, nouveau marqueur de progression tumorale et proteine diana de nouveaux composes antitumoraux
WO2001079293A2 (fr) * 2000-04-18 2001-10-25 Millennium Pharmaceuticals, Inc. 57809 et 57798, nouvelles molecules de cadherine humaines et utilisations
WO2003042409A2 (fr) * 2001-11-16 2003-05-22 Universita' Degli Studi Di Urbino Mutation de la ligne germinale dans le promoteur genique de la caderine e et methode de diagnostic d'identification d'une sensibilite accrue a la carcinome gastrique
WO2003097086A2 (fr) * 2002-05-15 2003-11-27 Technische Universität München Antagonistes de recepteur d'egf dans le traitement du cancer gastrique
US7569668B2 (en) 2002-02-20 2009-08-04 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Method to control tumor progression and invasiveness
WO2016030541A1 (fr) * 2014-08-29 2016-03-03 GAMBINO, Melania Pierina Procédé ex vivo pour la détection de marqueurs tumoraux dans un échantillon et ses utilisations

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* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, 15 July 1994, 54(14), BECKER K.F. et al., pages 3845-3852. *
CANCER RESEARCH, 15 September 1998, 58, GAYTHER S.A. et al., pages 4086-4089. *
CANCER, 15 September 1995, 76(6), YONEMURA Y. et al., pages 941-952. *
JAPANESE JOURNAL OF CANCER RESEARCH, August 1996, 87, MUTA H. et al., pages 843-848. *
JAPANESE JOURNAL OF CANCER RESEARCH, November 1996, 87, TAMURA G. et al., pages 1153-1159. *
JAPANESE JOURNAL OF CANCER RESEARCH, October 1994, 85, KANAI Y. et al., pages 1035-1039. *
JOURNAL OF ANALYTICAL CELLULAR PATHOLOGY, 1995, 8, YONEMURA et al., pages 177-190. *
LABORATORY INVESTIGATION, December 1996, 75(6), BECKER I. et al., pages 801-807. *
NATURE, 26 March 1998, 392(6674), GUILFORD P. et al., pages 402-405. *
PROC. NATL. ACAD. SCI. U.S.A., 1 March 1994, 91(5), ODA T. et al., pages 1858-1862. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7482159B2 (en) 1999-07-01 2009-01-27 Consejo Superior De Investigaciones Cientificas Snail, a new marker for tumour invasion and target protein of new antitumoral compounds
ES2161612A1 (es) * 1999-07-01 2001-12-01 Consejo Superior Investigacion Procedimiento para identificar un compuesto que inhiba la funcion represora de snail.
ES2161655A1 (es) * 1999-07-01 2001-12-01 Consejo Superior Investigacion Procedimiento para determinar la capacidad invasiva y metastasica de un tumor epitelial mediante el uso de snail.
US7482126B2 (en) 1999-07-01 2009-01-27 Consejo Superior De Investigacoines Cientificas Snail, a new marker for tumour invasion and target protein of new antitumoral compounds
WO2001002860A1 (fr) * 1999-07-01 2001-01-11 Consejo Superior De Investigaciones Cientificas Snail, nouveau marqueur de progression tumorale et proteine diana de nouveaux composes antitumoraux
US7794930B2 (en) 1999-07-01 2010-09-14 Consejo Superior De Investigaciones Cientificas Snail, a new marker for tumour invasion and target protein of new antitumoral compounds
WO2001079293A3 (fr) * 2000-04-18 2003-01-30 Millennium Pharm Inc 57809 et 57798, nouvelles molecules de cadherine humaines et utilisations
WO2001079293A2 (fr) * 2000-04-18 2001-10-25 Millennium Pharmaceuticals, Inc. 57809 et 57798, nouvelles molecules de cadherine humaines et utilisations
WO2003042409A2 (fr) * 2001-11-16 2003-05-22 Universita' Degli Studi Di Urbino Mutation de la ligne germinale dans le promoteur genique de la caderine e et methode de diagnostic d'identification d'une sensibilite accrue a la carcinome gastrique
WO2003042409A3 (fr) * 2001-11-16 2003-12-18 Univ Degli Studi Urbino Mutation de la ligne germinale dans le promoteur genique de la caderine e et methode de diagnostic d'identification d'une sensibilite accrue a la carcinome gastrique
US7569668B2 (en) 2002-02-20 2009-08-04 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Method to control tumor progression and invasiveness
WO2003097086A2 (fr) * 2002-05-15 2003-11-27 Technische Universität München Antagonistes de recepteur d'egf dans le traitement du cancer gastrique
WO2003097086A3 (fr) * 2002-05-15 2004-03-04 Univ Muenchen Tech Antagonistes de recepteur d'egf dans le traitement du cancer gastrique
WO2016030541A1 (fr) * 2014-08-29 2016-03-03 GAMBINO, Melania Pierina Procédé ex vivo pour la détection de marqueurs tumoraux dans un échantillon et ses utilisations

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