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WO1997015686A1 - Diagnostic de predisposition au cancer et traitement approprie - Google Patents

Diagnostic de predisposition au cancer et traitement approprie Download PDF

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Publication number
WO1997015686A1
WO1997015686A1 PCT/GB1996/002588 GB9602588W WO9715686A1 WO 1997015686 A1 WO1997015686 A1 WO 1997015686A1 GB 9602588 W GB9602588 W GB 9602588W WO 9715686 A1 WO9715686 A1 WO 9715686A1
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Prior art keywords
nucleic acid
dna
region
gene
markers
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PCT/GB1996/002588
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English (en)
Inventor
Nigel Kay Spurr
Ian Christopher Gray
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Imperial Cancer Research Technology Limited
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Application filed by Imperial Cancer Research Technology Limited filed Critical Imperial Cancer Research Technology Limited
Priority to KR1019980702948A priority Critical patent/KR19990067011A/ko
Priority to JP9516392A priority patent/JP2000500329A/ja
Priority to BR9611255-7A priority patent/BR9611255A/pt
Priority to AU73161/96A priority patent/AU722266B2/en
Priority to NZ320425A priority patent/NZ320425A/en
Priority to EP96935062A priority patent/EP0859860A1/fr
Publication of WO1997015686A1 publication Critical patent/WO1997015686A1/fr
Priority to US08/906,156 priority patent/US6287854B1/en
Priority to NO981662A priority patent/NO981662L/no

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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
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • 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
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates to methods of dete ⁇ iiining whether a patient has cancer or is susceptible to cancer, and it relates to methods of treating cancer, particularly prostate cancer.
  • Carcinoma of the prostate has become a most significant disease in many countries. Over the last 20 years the mortality rates have doubled and it is now the second commonest cause of male cancer deaths in England and
  • Prostate cancer screening currently consists of a rectal examination and measurement of prostate specific antigen (PSA) levels. These methods lack specificity as digital rectal examination has considerable inter- examiner variability (Smith & Catalona (1995) Urology 45, 70-74) and PSA levels may be elevated in benign prostatic hyperplasia (BPH), prostatic inflammation and other conditions.
  • BPH benign prostatic hyperplasia
  • the comparative failure of PSA as a diagnostic test was shown in 366 men who developed prostate cancer while being included in the Physicians Health Study, a prospective smdy of over 22,000 men. PSA levels were measured in serum, which was stored at the start of the study, and elevated levels were found in only 47% of men developing prostate cancer within the subsequent four years (Gann et al (1995) JAMA 273, 289-294).
  • SUBSTTTUTE SHEET (RULE 26) A number of tumours exhibit precise loss of the region 10q23-q25 (2, 3), suggesting the presence of a tumour suppressor gene in this area.
  • Mxil which encodes a negative regulator of the Myc oncoprotein and resides at 10q25, has been proposed as a candidate for the tumour suppressor gene (4); potentially disabling mutations of Mxil in a number of prostate tumours have recently been described.
  • Mxil displays allelic loss and mutation in some cases of prostate cancer and it has been concluded that it may contribute to the pathogenesis or neoplastic evolution of this common malignancy (5).
  • Objects of the invention are to provide better methods for the diagnosis of cancer and for determining susceptibility to cancer, especially prostate cancer; to provide nucleic acids which are useful in such methods; and to provide a tumour suppressor gene associated with prostate cancer.
  • SUBSTTTUTE SHEET (RULE 26 Furthermore, we failed to detect any mutations in Mx 7 in those tumours showing Mx/7-associated marker loss by either single-strand conformation polymorphism (SSCP) analysis or direct DNA sequencing, and our data indicate that Mxil is 20 centiMorgans away from the area of chromosome 10 that we have identified. We have found that all tumours which have a loss of lOq have loss of the region specified below.
  • SSCP single-strand conformation polymorphism
  • a first aspect of the invention provides a nucleic acid capable of selectively hybridising to the region of human chromosome 10 wliich region is bounded by DNA defmed by the markers D10S541 and D10S215 provided that the nucleic acid is not any one of the yeast artificial chromosomes (YACs) 746-H-8, 821-D-2, 831-E-5, 921-F-8, 738-B-12, 796-D-5, 829-E-l, 678-F-l, 839-B-l, 734-B-4, 7B-F12, 757-D-8, 773-C- 2, 787-D-7, 831-E-9, 855-D-2, 855-G-4, 876-G-ll, 894-H-5, 922-E-6, 934-D-3, 964- A-8, 968-E-6 or 24G-A10 and is not any one of the expressed sequence tags (ESTs) as described in Tables 3 to 22, and is
  • nucleic acid has sufficient nucleotide sequence similarity with the said chromosome 10 DNA that it can hybridise under moderately or highly stringent conditions.
  • stringency of nucleic acid hybridization depends on factors such as length of nucleic acid over which hybridisation occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and CG or AT content of the sequence.
  • Nucleic acids which can selectively hybridise to the said chromosome 10 DNA include nucleic acids which have > 95% sequence identity, preferably those with > 98% , more preferably those with > 99% sequence identity, over at least a portion of the nucleic acid with the said chromosome 10 DNA.
  • human genes usually contain introns such that, for example, a mRNA or cDNA derived from a gene within the said chromosome 10 DNA would not match perfectly along its entire length with the said chromosome 10 DNA but would nevertheless be a nucleic acid capable of selectively hybridising to the said region of chromosome 10.
  • Typical moderately or highly stringent hybridisation conditions which lead to selective hybridisation are known in the art, for example those described in Molecular Cloning, a laboratory manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
  • 20 x SSC may be prepared in the following way. Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H 2 O. Adjust the pH to 7.0 with a few drops of a 10 N solution of NaOH. Adjust the volume to 1 litre with H 2 O. Dispense into aliquots. Sterilize by autoclaving.
  • An example of a typical hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe is an oligonucleotide of between 15 and 50 bases is:
  • TMACl trimethylammonium chloride 0.01 M sodium phosphate
  • Tj is the irreversible melting temperamre of the hybrid formed between the probe and its target sequence. Jacobs et al (1988) Nucl. Acids Res. 16, 4637 discusses the dete ⁇ nination of TjS.
  • the recommended hybridization temperature for 17- mers in 3 M TMACl is 48-50°C; for 19-mers, it is 55-57°C; and for 20- mers, it is 58-66°C.
  • nucleic acid capable of selectively hybridising we also include nucleic acids which will amplify DNA from the said region of chromosome 10 by any of the well known amplification systems such as those described in more detail below, in particular the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Suitable conditions for PCR amplification include amplification in a suitable 1 x amplification buffer:
  • 10 x amplification buffer is 500 mM KCI; 100 mM Tris.Cl (pH 8.3 at room temperamre); 15 mM MgCl 2 ; 0.1 % gelatin.
  • the annealing part of the amplification is between 37 °C and 60°C, preferably 50°C.
  • the markers D10S541 and DS10S215 define regions on chromosome 10 which are indicated, for example, on the 1993-1994 Genethon human genetic linkage map which is described by Gyapay et al (1994) Nature Genetics 7, special issue No. 2, 246-339.
  • the aforementioned YACs are all publicly available from the CEPH mega- YAC library or the ICI YAC library (7B-F12 and 24G-A10), or from the Human Genome Mapping Project Resource Centre, Hinxton Hall, Hinxton, Cambridgeshire, CB10 IRQ, UK.
  • the position of the YACs on the genetic linkage map is made by reference to the CEPH- Genethon Quickmap database (Cohen et al (1993) Nature 366, 698-701).
  • an especially preferred nucleic acid of the invention is a nucleic acid capable of hybridising to the gene corresponding to the cDNA insert of clone IMAGE 264611.
  • IMAGE clone 264611 is publicly available from Research Genetics, Inc (2130 Memorial Parkway, SW Huntsville, AL 35801, USA) and other IMAGE sources eg American Type Culture Collection, Rockville, MD 20852, USA; Genome Systems Inc, 8629 Pennell Drive, St Louis, Missouri, MO 63114, USA, UK-HGMP Resource Centre, Hinxton, Cambridge CB10 1SB. The clone was obtained as described in the enclosed mformation for the ESTs N29304 and N20238 (see Tables 9 and 10). The clone is in a modified Pharmacia pT7T3 vector.
  • NAME pT7T3D-Pac (ampicillin resistant; 50 ⁇ g/ml)
  • V . TYPE plasmid
  • clones contain sequence that is part of the same gene as IMAGE clone 264611 since they overlap to form a largely contiguous sequence. All clones are freely available as physical entities unless otherwise noted. For each clone, some sequence, usually from the 5' or 3' ends, is available as ESTs which can be used to produce probes as described below.
  • the clones and their ESTs are listed on GenBank and the EMBL databases.
  • the nucleic acid is capable of selectively hybridising to the region of chromosome 10 bounded by DNA defined by the markers D10S541 and AFM337xf9.
  • Information on the marker AFM337xf9 is freely available from Genethon, 1 rue de LTnternationale, 91000 Evry, France.
  • AFM337xf9 is now known as D10S1765.
  • nucleic acid is capable of selectively hybridising to die human-derived DNA of any one of the YACs 746-H-8,
  • SUBSTTTUTE SHEET (RULE 26) 821-D-2, 831-E-5, 921-F-8, 796-D-5, 829-E-l, 839-B-l, 734-B-4 or 24G- A10; and it is still more preferred if the nucleic acid is capable of selectively hybridising to the human-derived DNA of any one of me YACs 746-H-8, 921-F-8, 821-D-2, 831-E-5, 796-D-5, 24G-A-10 or 734-B-4.
  • a YAC contains DNA which is required for propagation and maintenance in yeast.
  • the preferred nucleic acids of the invention are those that selectively hybridise to the human-derived DNA present in the YAC and not other DNA in the YAC, such as yeast DNA.
  • the human-derived cDNA insert of IMAGE clone 264611 hybridises to at least YAC clones 921F8, 746H8, 821D2, 831E5, 796D5 and 24GA10.
  • the human-derived cDNA insert of IMAGE clone 264611 hybridises to at least BAC (bacterial artificial chromosome) clones B2F20, B46B12, B60C5, B150K4, B150N3, B145C22, B181F15, and B188L22, but not to B76I10, B79A19, B7901, B93F12 and B122L22.
  • BAC bacterial artificial chromosome
  • the human-derived cDNA insert of IMAGE clone 264611 hybridises to at least PAC (Pl-derived artificial chromosome) clones P40F10 and P274D21, but not to P72G8, P74N2, P201J8, P201P5, P209K3 and P316N14.
  • PAC Pl-derived artificial chromosome
  • the PAC clones are publicly available from the Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 ISA, UK.
  • nucleic acid of the invention may be RNA or DNA, DNA
  • SUBSTTTUTE SHEET (RULE 26) is preferred.
  • nucleic acid of the invention may be double- stranded or single-stranded, single-stranded nucleic acid is preferred.
  • the nucleic acid of the invention may be very large, such as 100 kb, if it is double stranded. Indeed genes, such as a tumour suppressor gene, are often this large. However, for diagnostic, probing or amplifying purposes, it is preferred if the nucleic acid has fewer than 10 000, more preferably fewer than 1000, more preferably still from 10 to 100, and in further preference from 15 to 30 base pairs (if the nucleic acid is double- stranded) or bases (if the nucleic acid is single stranded). As is described more fully below, single-stranded DNA primers, suitable for use in a polymerase chain reaction, are particularly preferred.
  • An especially preferred nucleic acid of the invention is a nucleic acid capable of hybridising to the gene corresponding to the cDNA insert of clone IMAGE 264611 from which EST sequences N29304 and N20238 are derived.
  • the sequence and info ⁇ nation for N48030 and N20238 are recorded in the GenBank and EMBL databases (see Tables 9 and 12). Fragments and variants of this gene, and cDNAs derivable from the mRNA encoded by the gene are also preferred nucleic acids of the invention.
  • gene corresponding to the cDNA insert clone IMAGE 264611 we mean the gene which encodes mRNA which, when copied in part, produced the cDNA insert in said clone.
  • gene itself and variants and fragments thereof are a prefened nucleic acid of the invention.
  • gene we include not only the introns and exons but also regulatory regions associated with, and physically close to, the introns and exons, particularly those 5' to the 5'-most exon.
  • fragment of a gene we include any portion of the gene of at least 15
  • SUBSTTTUTE SHEET (RULE 26) nucleotides in length (whether single stranded or double stranded) but more preferably the fragment is at least 20 nucleotides in length, most preferably at least 50 nucleotides in length and may be at least 100 nucleotides in length or may be at least 500 nucleotides in length. Preferably the fragment is no more than 50 kb and, more preferably, no more than 100 kb.
  • variants of a gene we include specifically a cDNA, whether partial or full length, or whether copied from any splice variants of mRNA.
  • nucleic acid wherein, compared to me natural gene, nucleotide substitutions (including inversions), insertions and deletions are present whether in the gene or a fragment thereof or in a cDNA.
  • Both variants and fragments will be selected according to their intended purposes; for probing, amplifying or diagnostic purposes, shorter fragments but a greater degree of sequence identity (eg at least 80 % , 90 % , 95% or 99%) will generally be required than for the purposes of expressing a therapeutically useful product, where longer fragments will generally be needed but advantage can be taken of the redundancy in the genetic code, if desired.
  • a greater degree of sequence identity eg at least 80 % , 90 % , 95% or 99%
  • nucleic acid of the invention is an oligonucleotide primer which can be used to amplify a portion of the gene corresponding to the cDNA insert of clone IMAGE 264611.
  • nucleic acid of the invention comprises all or part of the gene and can be used as a probe for hybridisation.
  • FIG. 1 A cDNA sequence of IMAGE 264611 is shown in Figure 1.
  • SUBSTTTUTE SHEET (RULE 26) using methods well known in the art.
  • further cDNAs can be isolated from a prostate cDNA library using standard methods and the IMAGE 264611 clone as a probe or other probes readily derived from the sequences given in Tables 1 to 19 and the Figures. The sequence is readily determined using standard methods.
  • the gene can be isolated from a human genomic DNA library, using me IMAGE 264611 clone as a probe using standard methods or other probes readily derived from the sequences in Tables 1 to 19 and the Figures.
  • a prostate cDNA library may be obtained using standard molecular biology methods or may be obtained from Clontech Laboratories, Inc, 1020 East Meadow Circle, Palo Alto, California 94303-4230, USA.
  • the predicted amino acid sequence encoded by the IMAGE clone 264611 or the nucleotide sequences shown in Tables 3 to 22 may be used to make peptides which can, in turn, be used to make antibodies.
  • the antibodies can be used to screen a cDNA expression library or can be used to isolate the polypeptide encoded by the gene. Once the polypeptide is isolated its N-terminal sequence can be obtained using methods well known in the art.
  • the amino acid sequence is then used to design an oligonucleotide probe which identifies the 5' coding region of a cDNA.
  • SUBSTTTUTE SHEET (RULE 26) related approaches involve reverse transcription from mRNA using a primer based on the presently known 5' sequence which works back towards the 5' end of the mRNA transcript followed by PCR using random primers to prime from the "unknown" 5' end.
  • Messenger RNA- based RACE can also be used for obtaining 5' ends by isolating mRNA, removing the 5' cap and then the 5' end is ligated to an adaptor sequence and PCR follows using one primer against the adaptor and one primer specific to the cDNA of interest.
  • Vectorette PCR can be used for the identification of novel genes, or for the identification of additional sequence when part of the sequence of a gene is already known.
  • the vectorette itself is a double stranded piece of synthetic DNA, with a mismatched central region and one end suitable for ligation to DNA cut by a restriction enzyme (described in Current Protocols in Human Genetics 1995 (see pages 5.9.15-5.9.21) and in Valdes et al (1994) Proc. Natl. Acad. Sci. USA 91, 5377-5381 and Allen et al PCR Methods and Applications 4, 71-75).
  • PCR amplification is performed using a primer derived from the target DNA in conjunction with a primer derived from the mismatched region of the vectorette.
  • This vectorette primer has the same sequence as the bottom strand of this mismatched region and therefore has no complementary sequence to anneal to in the first cycle of PCR.
  • the first round of amplification is unidirectional, as priming can only occur from
  • SUBSTTTUTE SHEET (RULE 26) the primer within the target DNA. This produces a complementary strand for the vectorette PCR primer to anneal to in the second PCR cycle. In the second and subsequent cycles of PCR, bodi primers can prime DNA synthesis with the end result being that the only fragment amplified contains the sequence of interest.
  • This technique can be used for the identification of intronic sequences within a gene based on a knowledge of the cDNA sequence for that gene.
  • a primer designed from the cDNA sequence is used in conjunction with the vectorette primer to PCR amplify a specific fragment of the gene. Exon/intron boundaries can be identified by comparison of the sequence of this fragment to that of the cDNA.
  • This method has been used in combination with primers derived from cDNA clone 264611 to identify intron sequences (see Figures 8-15).
  • a vectorette approach can be used to identify the missing 5 ' end of a gene by using a primer derived from the 5' end of the known cDNA sequence to generate further 5' sequence data.
  • Vectorettes can also be used for the identification of completely novel gene sequences in a technique known as 'island rescue'. This approach exploits the fact that CpG-rich 'islands' exist within mammalian genomes and that such islands are associated with the 5' ends of genes. Certain restriction enzymes cut within CpG islands, for example, the enzyme N ⁇ fl. Following NotI digestion of a genomic D ⁇ A fragment, a vectorette with a N ⁇ fl-compatible sticky end is ligated to the resulting sub-fragments. PCR amplification is then performed using the vectorette primer in conjunction with a primer derived from an Alu repeat element.
  • polypeptide encoded by the gene corresponding to the cDNA clone IMAGE 264611 or the nucleotide sequences shown in Tables 3 to 22 has some sequence similarity to the polypeptide tensin, a protein involved in cytoskeletal/extracellular matrix interactions; similarity is also observed, at least at the nucleotide sequence level, with auxilin, a protein involved in protein transport to the cell membrane via clathrin coated vesicles. Sequence similarity between tensin and auxilin has also been noted previously.
  • a preferred nucleic acid of the invention is one comprising a tumour suppressor gene or fragment or variant thereof.
  • the tumour suppressor gene is one which is involved in the origin or development of a cancer such as prostate cancer, melanoma, glioma or non-Hodgkin's lymphoma.
  • the tumour suppressor gene is involved in the origin or development of prostate cancer, particularly prostate adenocarcinoma.
  • a nucleic acid of the invention comprising a tumour suppressor gene or fragment or derivative thereof is readily identified; for example, the gene may be identified by screening a panel of RNAs from prostate and other tumour cell lines in order to identify a reduced level of transcript.
  • the transcript may be large, as it will probably have a complex function and several sites for disabling mutation 'hits' (as is the case with the tumour
  • SUBSTTTUTE SHEET (RULE 26) suppressor genes BRCA1, RB).
  • Cross-species conservation indicates that the gene has a basic cell 'housekeeping' function, the loss of which may lead to loss of growth control and tumour formation.
  • tumor suppressor gene we include any gene for which loss or some reduction in any of its function or activities can contribute to neoplasia.
  • SSCP single-stranded conformation polymorphism
  • tumour suppressor gene of the invention almost certainly contains introns (as does the gene corresponding to IMAGE clone 264611) and almost certainly is >0.5 kb, more likely > 1.0 kb and most likely between 1.0 kb and 500 kb.
  • the cDNA insert in IMAGE clone 264611 is about 1.7 kbp.
  • Any tumour suppressor gene of the invention almost certainly is polymorphic in its DNA sequence.
  • fragments such as restriction fragments or fragments derived by enzymatic amplification
  • variants such as natural variants, eg allelic variants
  • variants created by in vitro manipulation are part of the invention.
  • Suitable such fragments include fragments which are useful as a hybridisation probe or fragments which are useful as an amplification primer.
  • Suitable such variants include variants in which the coding sense of the gene is unaltered or variants in which the coding sequence is modified so as to alter the properties of the encoded polypeptide.
  • tumour suppressor gene of the invention almost certainly ultimately encodes a polypeptide, it may encode an RNA species which RNA species does not encode a polypeptide.
  • the nucleic acid comprises a nucleic acid product of a tumour suppressor gene or derivative or fragment or variant thereof.
  • nucleic acids include mRNA transcribed from the tumour suppressor gene.
  • the nucleic acid is a cDNA (copy DNA) derived from a mRNA transcribed from the tumour suppressor gene.
  • cDNA copy DNA
  • Libraries of cDNA derived from selected tissues, such as prostate or prostate tumour tissue, are known in the art and can be prepared from suitable mRNA using methods known in the art for example as described in Molecular cloning, a laboratory manual (supra).
  • nucleotide sequences described in Tables 3 to 22 are partial sequences of partial cDNAs the said cDNAs being derived from mRNAs which are related to, selectively hybridise to, and are almost certainly transcribed from a gene or genes found in the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215.
  • the nucleotide sequences shown in Figures 8 to 15 include sequences from introns in the gene corresponding to IMAGE clone 264611.
  • SUBSTTTUTE SHEET represent the mRNA products of at least one gene which is found within the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215; more particularly in the sub-region defined by the YAC clones.
  • a particularly preferred embodiment comprises a nucleic acid capable of selectively hybridising to me region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215 and capable of selectively hybridising to the human-derived sequence as described in any one of Tables 3 to 22 and Figures 6 and 8 to 15 provided that the nucleic acid is not any one of the yeast artificial chromosomes (YACs) 746-H-8, 821-D- 2, 831-E-5, 921-F-8, 738-B-12, 796-D-5, 829-E-l, 678-F-l, 839-B-l , 734-B-4, 7B-F12, 757-D-8, 773-C-2, 787-D-7, 829-E-l, 831-E-9, 855-D- 2, 855-G-4, 876-G-ll , 894-H-5, 921-F-8, 922-E-6, 934-D-3, 964
  • the nucleic acid comprises the gene or genes from which the sequence of any one of Tables 3 to 22 and Figures 6 and 8 to 15 is derived or a fragment or variant thereof. It is also preferred if the nucleic acid comprises a full length cDNA or a cDNA which is at least 50% of the length of a mRNA transcript; more preferably greater than 75% of the length; more preferably greater than 95% of the length.
  • SUBSTTTUTE SHEET (RULE 26) It may be desirable to subclone the nucleic acid, particularly if all or part of the protein coding sequence is to be expressed.
  • die DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • bacteria for example E. coli and Bacillus subtilis
  • yeasts for example Saccharomyces cerevisiae
  • filamentous fungi for example Aspergillus
  • plant cells animal cells and insect cells.
  • the vectors include a prokaryotic replicon, such as the ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression
  • the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
  • an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical prokaryotic vector plasmids are pUCl ⁇ , pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and p2 c99A andpKK223-3 available from Pharmacia, Piscataway, NJ, USA.
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
  • an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRPl, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Integrating plasmids
  • a variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
  • the DNA segment generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded teimini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends wim their polymerizing activities.
  • the combination of these activities therefore generates blunt-ended DNA segments.
  • the blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying polymeric linker sequences at their ends.
  • These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
  • nucleic acids of the first aspect of the invention are those selected from the group consisting of primers suitable for amplifying nucleic acid.
  • me nucleic acids are selected from the group consisting of primers which hybridise to the nucleotide sequences as described in any one of Tables 3 to 22 and Figures 6 and 8 to 15, or their complement.
  • the amplification primers hybridise to the introns of a gene. They are particularly useful if processed pseudogenes are present.
  • the nucleic acids are selected from the group consisting of primers which hybridise to the sequences given in Figures 6 and 8 to 15, or their complement.
  • PCR polymerase chain reaction
  • the PCR primers do not contain any complementary strucmres with each other longer than 2 bases, especially at their 3' ends, as this feamre 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”.
  • SUBSTTTUTE SHEET (RULE 26) calculations used in conjunction with DNA probe hybridization studies often predict that a given primer should anneal at a specific temperature or that the 72° C extension temperature will dissociate the primer/template hybrid prematurely. In practice, the hybrids are more effective in the PCR process than generally predicted by simple T m calculations.
  • Optimum annealing temperatures may be determined empirically and may be higher than predicted.
  • Taq 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.
  • nucleic acid amplification protocols can be used in the method of the invention including the polymerase chain reaction, QB replicase and ligase chain reaction.
  • NASBA nucleic acid sequence based amplification
  • 3SR can be used as described in Compton (1991) Nature 350, 91-92 and AIDS (1993)
  • Vol 7 (Suppl 2)
  • S108 or SDA strand displacement amplification
  • the polymerase chain reaction is particularly preferred because of its simplicity.
  • oligonucleotide probe hybridises to the interprimer sequence as defined by me 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.
  • a radionuclide such as 32 P, 33 P and 35 S
  • 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 electrochemilu ⁇ tinescence-based detection system for automated PCR product quantitation" BioTechniques 15, 152-157.
  • PCR products can also be detected using a probe which may have a fluorophore-quencher pair or may be attached to a solid support or may have a biotin tag or they may be detected using a combination of a capture probe and a detector probe.
  • Fluorophore-quencher pairs are particularly suited to quantitative measurements of PCR reactions (eg RT-PCR). Fluorescence polarisation using a suitable probe may also be used to detect PCR products.
  • nucleic acids are those which will act as PCR primers which primers can be selected by reference to the sequence shown in Figures 6 and 8 to 15. These primers are useful in amplifying DNA derived from the gene corresponding to the cDNA clone IMAGE 264611. These primers include, but are not limited to, the sequences which are given on Figures 8 to 15 in bold (see Figure legends). The downstream (3') primers are the reverse complement of the sequences indicated in bold.
  • Oligonucleotide primers can be synthesised using methods well known in the art, for example using solid-phase phosphoramidite chemistry.
  • a second aspect of the invention provides a nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defmed by the markers D10S541 and D10S215, further comprising a detectable label.
  • detecttable label we include any convenient radioactive label such as 32 P, 33 P or 35 S which can readily be incorporated into a nucleic acid molecule using well known methods; we also include any convenient fluorescent or chemiluminescent label which can readily be incorporated into a nucleic acid.
  • detecttable label also includes a moiety which can be detected by virtue of binding to another moiety (such as biotin which can be detected by binding to streptavidin); and a moiety, such as an enzyme, which can be detected by virtue of its ability to convert a colourless compound into a coloured compound, or vice versa (for example, alkaline phosphatase can convert colourless o- nitrophenylphosphate into coloured o-nitrophenol).
  • the nucleic acid probe may occupy a certain position in a fixed assay and whether the nucleic acid hybridises to the said region of human chromosome 10 can be determined by reference to the position of hybridisation in the fixed assay.
  • the detectable label may also be a fluorophore-quencher pair as described in Tyagi & Kramer (1996) Nature Biotechnology 14, 303-308.
  • nucleic acid comprises the human-derived sequence in any one of the expressed sequence tags (ESTs) as described in Tables 3 to 22 or the cDNA described in Figure 6 or the intron sequences shown in Figures 8 to 15 further comprising a detectable label; or if the nucleic acid comprises the human-derived sequence in any one of the yeast
  • SUBSTTTUTE SHEET (RULE 26) artificial chromosomes (YACs) 921-F-8, 746-H-8, 821-D-2, 831-E-5, 796- D-5, 24G-A-10 or 734-B-4 or BAC clones B2F20, B46B12, B60C5, B150K4, B150N3, B145C22, B181F15, B188L22, or PAC clones P40F10, and P274D21.
  • YACs artificial chromosomes 921-F-8, 746-H-8, 821-D-2, 831-E-5, 796- D-5, 24G-A-10 or 734-B-4 or BAC clones B2F20, B46B12, B60C5, B150K4, B150N3, B145C22, B181F15, B188L22, or PAC clones P40F10, and P274D21.
  • nucleic acids are diose of the first aspect of the invention further comprising a detectable label.
  • a third aspect of the invention provides a method for determining the susceptibility of a patient to cancer comprising the steps (i) obtaining a sample containing nucleic acid derived from the patient; and (ii) contacting the said nucleic acid with a nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215.
  • the method is suitable for determining the susceptibility of a patient to any cancer but it is prefened if the cancer for which susceptibility is determined is prostate cancer, melanoma, glioma or non-Hodgkin's lymphoma.
  • the method is most suited for determining the susceptibility of a patient to prostate cancer. Accordingly, at least for the dete ⁇ nination of susceptibility to prostate cancer, the patient is male.
  • the presence or absence of a portion of human chromosome 10 may be determined by the methods of the third, fourth and fifth aspects of the invention, and in a preferred embodiment of the third, fourth and fifth aspects of the invention me nucleic acid capable of selectively hybridising to the said region of human chromosome 10 is a nucleic acid suitable for amplification of a portion of the said region of chromosome 10.
  • a fourth aspect of the invention provides a method of diagnosing cancer in a patient comprising the steps of (i) obtaining a sample containing nucleic acid derived from the patient; and (ii) contacting the said nucleic acid witii a nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215.
  • the method is particularly suited for distinguishing between neoplasia and hyperplasia of the prostate. Because all tumours which have a loss of lOq have also been found to lack the region specified herein, a differential diagnostic test can be performed, using the markers of the invention and other markers (including markers on other chromosomes).
  • a fifth aspect of the invention provides a metiiod of predicting the relative prospects of a particular outcome of a cancer in a patient comprising the steps of (i) obtaining a sample containing nucleic acid derived from the patient; and (ii) contacting the said nucleic acid with a nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215.
  • any sample containing nucleic acid derived from the patient is useful in the methods of the third, fourth and fifth aspects of the invention, it is prefened if the sample is selected from the group consisting of prostate tissue, blood, urine or semen.
  • Prostate tissue can be obtained from a patient using standard surgical techniques. Cells derived from the prostate are found in small numbers in the urine and in the blood.
  • the sample containing nucleic acid from the patient is, or is derived directly from, a cell of the patient, such as a prostate cell
  • a sample indirectly derived from a patient such as a cell grown in culmre, is also included within the invention. Equally, although
  • the nucleic acid derived from the patient may have been physically within the patient, it may alternatively have been copied from nucleic acid which was physically within the patient.
  • the tumour tissue may be taken from the primary tumour or from metastases, and particularly may be taken from the margins of the tumour.
  • the nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215 further comprises a detectable label.
  • the detectable label includes the labels described above in relation to the second aspect of the invention.
  • the aforementioned methods may be used for presymptomatic screening of a patient who is in a risk group for cancer. For example, men older than about 60 years are at greater risk of prostate cancer than men below the age of 35. Similarly, the methods may be used for the pathological classification of tumours such as prostate tumours.
  • nucleic acid which is capable of the said selective hybridisation is contacted with a nucleic acid derived from the patient under hybridising conditions.
  • Suitable hybridising conditions include those described in relation to the first aspect of the invention.
  • the source of the said sample containing nucleic acid derived from the patient that the sample is enriched for prostate-derived tissue or cells.
  • Enrichment for prostate cells may be achieved using, for example, cell sorting methods such as fluorescent activated cell sorting (FACS) using a prostate-selective antibody such as one directed to prostate-specific antigen (PSA).
  • FACS fluorescent activated cell sorting
  • PSA prostate-specific antigen
  • the source of the said sample also includes biopsy material and tumour samples, also including fixed paraffin mounted specimens as well as fresh or frozen tissue.
  • the methods of the third, fourth or fifth aspect of the invention may involve sequencing of DNA at one or more ofthe relevant positions within the relevant region, including direct sequencing; direct sequencing of PCR-amplified exons; differential hybridisation of an oligonucleotide probe designed to hybridise at the relevant positions within the relevant region (conveniently this uses immobilised oligonucleotide probes in, so- called, "chip” systems which are well known in the art); denaturing gel electrophoresis following digestion with an appropriate restriction enzyme, preferably following amplification of the relevant DNA regions; SI nuclease sequence analysis; non-denaturing gel electrophoresis, preferably following amplification ofthe relevant DNA regions; conventional RFLP (restriction fragment length polymo ⁇ hism) assays; heteroduplex analysis; selective DNA amplification using oligonucleotides; fluorescent in-situ hybridisation of interphase chromosomes; ARMS-PCR (Amplification Refractory Mutation System-PCR) for specific mutation
  • SUBSTTTUTE SHEET (RULE 26) is commercially available from GibcoBRL, Life Technologies, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, Scotland.
  • PCR amplification of smaller regions to detect small changes greater than 3-4 bp insertions or deletions may be prefened.
  • Amplified sequence may be analysed on a sequencing gel, and small changes (minimum size 3-4 bp) can be visualised.
  • Suitable primers are designed as herein described.
  • genomic DNA restriction enzyme digestion, gel electrophoresis, Southern blotting, and hybridisation specific probe (any of the YACs, BACs, in the region as described herein, or a suitable fragment derived therefrom).
  • PCR amplify DNA, restriction enzyme digestion, gel detection by ethidium bromide, silver staining or inco ⁇ oration of radionucleotide or fluorescent primer in the PCR.
  • RNA and cDNA for prostate specific tissue.
  • ASOs allele specific oligonucleotides
  • the method also includes checking for loss-of-heterozygosity (LOH; shows one copy lost) and then look for loss of function of RNA by failing to detect a mRNA on Northern blots or by PCR or in RNA/cDNA (shows other copy non-active).
  • LOH on a tumour cell, from whatever source, compared to blood is useful as a diagnostic tool, eg show that the tumour has progressed and requires more stringent treatment.
  • the nucleic acid is capable of selectively hybridising to the region of human chromosome 10 which region is bounded by the markers D10S541 and D10S215; more preferably the said nucleic acid comprises or is capable of selectively hybridising to the human-derived DNA of any one of YACs 746-H-8, 821-D-2, 831-E-5, 921-F-8, 796-D-5, 829-E-l , 839-B-l, 734-B-
  • the nucleic acid comprises or is capable of selectively hybridising to the human-derived DNA of any one of the YACs 821-D-2, 831-E-5, 796-D-5, 24G-A-10 or 734-B-4.
  • the nucleic acid comprises or is capable of selectively hybridising to the human-derived DNA of any of the BACs or PACs B2F20, P40F10, P72G8, P74N2, P274D21, B76I10, B79A19, B7901, B93F12, B122L22, P201J8, P201P5, P209K3, P316N14, B46B12, B60C5, B145C22, B150K4, B150N3, B181F15, and B188L22.
  • nucleic acid is a primer for the microsatellite markers D10S541 , D10S215 and AFM337xf9 (D10S1765), namely:
  • nucleic acid is capable of selectively hybridising to the gene conesponding to the cDNA insert of the clone IMAGE 264611.
  • the present invention provides a use of a nucleic acid which is capable of selectively hybridising to the said region of human chromosome 10 in diagnosing cancer or diagnosing susceptibility to cancer.
  • the present invention provides a method of deterrnining the presence or absence, or a mutation in, the said region of human chromosome 10.
  • the said nucleic acid capable of selectively hybridising is DNA, and also preferably the said nucleic acid is single-stranded.
  • the said nucleic acid capable of selectively hybridising has fewer than 10 000 base pairs when the nucleic acid is double-stranded or bases when the nucleic acid is single-stranded; more preferably if the said nucleic acid has fewer than 1000 base pairs when the nucleic acid is double-stranded or bases when the nucleic acid is single- stranded; more preferably still if the said nucleic acid has from 10 to 100 base pairs when the nucleic acid is double-stranded or bases when the nucleic acid is single-stranded; and even more preferably if the said nucleic acid has from 15 to 30 base pairs when the nucleic acid is double- stranded or bases when the nucleic acid is single-stranded.
  • SUBSTTTUTE SHEET (RULE 26) It is prefened if the said nucleic acid capable of selectively hybridising comprises a tumour suppressor gene or fragment or variant thereof, or a nucleic acid which selectively hybridises thereto.
  • Suitable primers include those described in relation to the first and second aspects of the invention.
  • reverse transcriptase PCR is used to detect micrometastases in blood samples from the patient.
  • a blood sample is taken and RNA prepared from the nucleated cells in the sample.
  • This is used in PCR amplification with oligonucleotide primers detecting the presence or absence, or mutations in prostate tumour suppressor mRNA.
  • This is a relatively sensitive method that can detect one cell in a mix of more than a million normal cells and it is possible to detect prostate tumour suppressor mRNA products present in circulating metastatic cells mixed with normal blood cells that do not express these genes.
  • the gene products of those genes present in the region of chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215, are useful markers detecting circulating prostate cells.
  • micrometastases by looking for mutations in the DNA of cells in the blood sample directly, or by using the protein truncation test or by using microsatellite markers; in this case the suspected tumour cells should be purified from the blood.
  • the said nucleic acid capable of selectively hybridising is, or is capable of hybridising to, the human derived sequence as described in Tables 3 to 22 or Figures 6 and 8 to 15; conveniently the said nucleic acid is selected from the group consisting of primers which hybridise to DNA from the sequences as described in Tables 3 to 22 or Figures 6 and 8 to 15.
  • the methods of the invention include the detection of mutations in the region of chromosome 10 bounded by DNA defined by the markers D10S541 and D10S215; especially in the tumour suppressor gene.
  • the methods of the invention may make use of a difference in restriction enzyme cleavage sites caused by mutation.
  • a non-denaturing gel may be used to detect differing lengths of fragments resulting from digestion with an appropriate restriction enzyme.
  • the DNA is usually amplified before digestion, for example using the polymerase chain reaction (PCR) method and modifications thereof. Otherwise 10-100 times more DNA would need to be obtained in the first place, and even then the assay would work only if the restriction enzyme cuts DNA infrequentiy.
  • PCR polymerase chain reaction
  • Amplification of DNA may be achieved by the established PCR method as disclosed by Saiki et al (1988) Science 239, 487-491 or by developments thereof or alternatives such as the ligase chain reaction, QB replicase and nucleic acid sequence-based amplification or other known amplification methods, some of which are described herein.
  • an "appropriate restriction enzyme” is one which will recognise and cut the wild-type sequence and not the mutated sequence or vice versa.
  • the sequence which is recognised and cut by the restriction enzyme can be present as a consequence of the mutation or it can be introduced into the normal or mutant allele using mismatched oligonucleotides in the PCR reaction. It is convenient if the enzyme cuts DNA only infrequentiy, in other words if it recognises a sequence which occurs only rarely.
  • a pair of PCR primers are used wliich match (ie hybridise to) either the wild-type genotype or the mutant genotype but not both. Whether amplified DNA is produced will then indicate the wild- type or mutant genotype (and hence phenotype).
  • this method relies partly on a negative result (ie the absence of amplified DNA) which could be due to a technical failure. It is therefore less reliable and/or requires additional control experiments.
  • a preferable method employs similar PCR primers but, as well as hybridising to only one of the wild-type or mutant sequences, they introduce a restriction site which is not otherwise there in either the wild- type or mutant sequences.
  • the nucleic acids provided by the present invention are useful for a number of pu ⁇ oses. They can be used in Southern hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above.
  • the probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the tumour suppressor gene or mRNA using other techniques. Mismatches can be detected using either enzymes (eg SI nuclease or resolvase), chemicals (eg hydroxylamine or osmium tetroxide and piperidine), or changes in electrophoretic mobility of mismatched hybrids as compared to totally matched hybrids. These techniques are known in the art.
  • the probes are complementary to the tumour suppressor gene coding sequences, although probes to certain introns are also contemplated.
  • An entire battery of nucleic acid probes may be used to compose a kit for detecting loss of or mutation in wild-type tumour suppressor genes.
  • the kit allows for hybridization to the entire mmour suppressor gene.
  • the probes may overlap with each other or be contiguous.
  • a riboprobe is used to detect mismatches with mRNA, it is complementary to the mRNA of the human wild-type tumour suppressor gene.
  • the riboprobe thus is an anti-sense probe in that it does not code for the protein encoded by the tumour suppressor gene because it is of the opposite polarity to the sense strand.
  • the riboprobe generally will be labelled, for example, radioactively labelled which can be accomplished by any means known in the art. If the riboprobe is used to detect mismatches with DNA it can be of either polarity, sense or anti-sense. Similarly, DNA probes also may be used to detect mismatches.
  • Nucleic acid probes may also be complementary to mutant alleles of the tumour suppressor gene. These are useful to detect similar mutations in other patients on the basis of hybridization rather than mismatches.
  • the tumour suppressor gene probes can also be used in Southern hybridizations to genomic DNA to detect gross chromosomal changes such as deletions and insertions.
  • the probes can also be used to select cDNA clones of mmour suppressor genes from tumour and normal tissues.
  • the probes can be used to detect tumour suppressor gene mRNA in tissue to determine if expression is altered, for example diminished, as a result of loss of wild-type mmour suppressor genes.
  • loss of the wild-type gene is detected.
  • the loss may be due to either insertional, deletional or point mutational events. If only a single allele is mutated, an early neoplastic state may be indicated. However, if both alleles are mutated then a malignant state is indicated or an increased probability of malignancy is indicated.
  • a tumour suppressor gene allele which is not deleted eg that on the sister chromosome to a chromosome carrying a gene deletion
  • Mutational 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 mmour suppressor gene product.
  • the invention also includes the following methods: in vitro transcription and translation of tumour suppressor gene to identify truncated gene products, or altered properties such as substrate binding; immunohistochemistry of tissue sections to identify cells in which expression of the protein is reduced/lost, or its distribution is altered within cells or on their surface; and the use of RT-PCR using random primers, prior to detection of mutations in the region as described above.
  • a sixth aspect of the invention provides a system (or it could also be termed a kit of parts) for detecting the presence or absence of, or mutation in, the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215, the system comprising a nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215 and a nucleoside triphosphate or deoxynucleoside triphosphate or derivative thereof.
  • Prefened nucleic acids capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by markers D10S541 and D10S215 are the same as those prefened in the third, fourth and fifth aspects of the invention.
  • mutation we include insertions, substitutions and deletions.
  • nucleoside triphosphate or deoxynucleoside triphosphate or derivative thereof we include any naturally occuning nucleoside triphosphate or deoxynucleoside triophosphate such as ATP, GTP, CTP, and UTP, dATP dGTP, dCTP, TTP as well as non-naturally derivatives such as those that include a phosphorothioate linkage (for example ⁇ S derivatives).
  • nucleoside triphosphate or deoxynucleoside triphosphosphate is radioactively labelled or derivative thereof, for example with 32 P, 33 P or 35 S, or is fluorescently labelled or labelled with a chemiluminescence compound or with digoxygenin.
  • deoxynucleotides are at a concentration suitable for dilution to use in a PCR.
  • the invention includes a kit of parts which includes a nucleic acid capable of selectively hybridising to the said region of human chromosome 10 and means for detecting the presence or absence of, or a mutation in, the said region.
  • a seventh aspect of the invention provides a system for detecting the presence or absence of, or mutation in, the region of human chromosome
  • nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215 and a nucleic acid modifying enzyme.
  • Prefened nucleic acids capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by markers D 10S541 and D 10S215 are the same as those prefened in the third, fourth and fifth aspects of the invention.
  • mutation we include insertions, substitutions (including transversions) and deletions.
  • nucleic acid modifying enzyme we include any enzyme capable of modifying an RNA or DNA molecule.
  • Prefened enzymes are selected from the group consisting of DNA polymerases, DNA ligases, polynucleotide kinases or restriction endonucleases.
  • a particularly prefened enzyme is a thermostable DNA polymerase such as Taq DNA polymerase.
  • Nucleases such as Cleavase I which recognise secondary structure, for example mismatches, may also be useful.
  • An eighth aspect of the invention provides a polypeptide capable of being encoded by the tumour suppressor gene of the invention or a fragment or variant thereof.
  • the polypeptide preferably has tumour suppressor activity, especially in the prostate, or cross-reacts with an antibody which is specific for the native polypeptide.
  • a ninth aspect of the invention comprises a molecule capable of specifically binding with a polypeptide of the eighth aspect of the invention.
  • the molecule is an antibody-like molecule comprising
  • SUBSTTTUTE SHEET (RULE 26) complementarity-determining regions specific for the said polypeptide.
  • Monoclonal antibodies which will bind to many of these antigens are already known but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies can be prepared to most antigens.
  • the antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]).
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques " , H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hunell (CRC Press, 1982).
  • Non-human antibodies can be "humanized” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
  • Further aspects of the invention provide methods (a) for determining the susceptibility of a patient to cancer comprising the steps of (i) obtaining a sample containing protein derived from the patient; and (ii) deteimining the relative amount or size in the said sample of the polypeptide according to the eighth aspect of the invention or dete ⁇ nining whether there is a truncation of, or loss of function of, a polypeptide according to the eighth aspect of the invention; (b) of diagnosing cancer in a patient comprising the steps of (i) obtaining a sample containing protein derived from the patient; and (ii) determining the relative amount or size in the said sample of the polypeptide according to the eighth aspect of the invention; and (c)
  • SUBSTTTUTE SHEET (RULE 26) of predicting the relative prospects of a particular outcome of a cancer in a patient comprising the steps of (i) obtaining a sample containing protein derived from the patient; and (ii) deterrnining the relative amount in the said sample of the polypeptide according to the seventh aspect of the invention.
  • the protein in the cancer cell is truncated or the amount of protein product is decreased.
  • derived from the patient we include a sample directly derived from the patient or indirectly derived from, for example the protein may be produced from isolated DNA from the patient by in vitro transcription and translation.
  • the sample may be any suitable sample and includes biopsy material, tumour samples (for example, those on fixed paraffin mounts and fresh and frozen tissue) and cells shed from mmour samples.
  • These methods are suited to determining the susceptibility of a patient to any cancer but are particularly suited to prostate cancer, melanoma, glioma or non-Hodgkin's lymphoma. Accordingly, at least for the determination of susceptibility to prostate cancer, the patient is male. Prostate cancer is particularly relevant.
  • the said polypeptide is detected using a molecule as defined in the ninth aspect of the invention.
  • the molecule is an antibody-like molecule comprising complementarity-deter ⁇ iining regions specific for the polypeptide.
  • the molecule such as a monoclonal antibody, comprises a detectable label.
  • Suitable detectable labels include radioactive labels such as 125 I and 13, I and other radionuclides such as those used in diagnostic imaging, as well as any convenient fluorescent or chemiluminescent label which can readily be inco ⁇ orated into the molecule, such as an antibody.
  • detectable label also includes a moiety which can be detected by virtue of binding to another moiety (such as biotin which can be detected by binding to streptavidin); and a moiety, such as an enzyme, which can be detected by virtue of its ability to convert a colourless compound into a coloured compound, or vice versa (for example, alkaline phosphalase can convert colourless o-nitrophenylphosphate into coloured o-nitrophenol).
  • the antibodies are raised to peptides encoded by different exons of the said polypeptide. These can be used to detect truncated proteins, for example in tissue sections, as well as in protein truncation assays, and can also be used to detect changes in the level of proteins.
  • a further aspect of the invention provides the use of a nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215 in the manufacture of a reagent for diagnosing cancer, especially prostate cancer; and in the manufacmre of a medicament for treating cancer.
  • a still further aspect of the invention provides a method of treating cancer comprising the step of administering to the patient a nucleic acid capable of selectively hybridising to the region of human chromosome 10 which region is bounded by DNA defined by the markers D10S541 and D10S215, the nucleic acid encoding, optionally when inserted into the patient, a tumour-suppressing molecule.
  • Tumour suppression may be identified by transfecting a (preferably prostate) tumour cell line with an expression vector comprising the polynucleotide and comparing the tumorigenic properties of the transfected cell line with the parental line in a xenograft model (eg nude mice).
  • the method is for treating prostate cancer.
  • the nucleic acid is a tumour suppressor gene which, in this context, is a therapeutic gene.
  • the wild-type tumour suppressor gene is prefened.
  • the nucleic acid comprises a suitable delivery system.
  • retrovirus derived vectors are suited for the repair of gene defects in resting or slowly dividing tissue cells
  • retrovirus derived vectors specifically target rapidly dividing cells (eg tumour cells) and are therefore suited for the in vivo treatment of cancer therapies.
  • viral vectors capable of cellular gene integration may be more beneficial than non-integrating alternatives (eg adenovirus derived vectors) when repeated therapy is undesirable for immunogenicity reasons.
  • the highest level of expression is likely to be achieved using viral promoters, for example, the Rous sarcoma virus long terminal repeat (Ragot et al (1993) Nature 361, 647-650; Hyde et al (1993) Nature 362, 250-255) and the adenovirus major late promoter.
  • the latter has been used successfully to drive the expression of a cystic fibrosis transmembrane conductance regulator (CFTR) gene in lung epithelium (Rosenfeld et al (1992) Cell 68, 143-155). Since these promoters function in a broad range of tissues they may not be suitable to direct cell-type-specific expression unless the delivery method can be adapted to provide the specificity.
  • somatic enhancer sequences could be used to give cell-type-specific expression in an extrachromosomal setting.
  • SUBSTTTUTE SHEET (RULE 26) Where withdrawal of the gene- vector construct is not possible, it may be necessary to add a suicide gene to the system to abort toxic reactions rapidly.
  • the he ⁇ es simplex virus thymidine kinase gene when transduced into cells, renders them sensitive to the drug ganciclovir, creating the option of killing the cells quickly.
  • ectotropic viruses which are species specific, may provide a safer alternative to the use of amphotropic viruses as vectors in gene therapy.
  • a human homologue of the non-human, ectotropic viral receptor is modified in such a way so as to allow recognition by the virus.
  • the modified receptor is then delivered to cells by constructing a molecule, the front end of which is specified for the targeted cells and the tail part being the altered receptor.
  • the genetically engineered ectotropic virus, carrying the therapeutic gene can be injected and will only integrate into the targeted cells.
  • Virus-derived gene transfer vectors can be adapted to recognize only specific cells so it may be possible to target the cancer cell, such as prostate mmour cell. Similarly, it is possible to target expression of the therapeutic gene to the cancer cell, particularly prostate cell, using a prostate-specific promoter such as that for the PSA gene.
  • a further aspect of the invention provides a method of treating cancer comprising the step of administering a molecule according to the ninth aspect ofthe invention to the patient, the said molecule further comprising a cytotoxic moiety.
  • the cytotoxic moiety may be directly cytotoxic (such as ricin, a suitable drug or suitable radionuclide) or it may be indirectly cytotoxic (such as an enzyme which is capable of converting a relatively non-toxic pro-drug into a relatively toxic drug; see for example WO
  • the molecule according to the ninth aspect of the invention is an antibody, preferably monoclonal antibody, or fragment thereof.
  • the aforementioned compounds of the invention or a formulation thereof may be administered by parenteral (eg subcutaneous or intramuscular) injection but preferably into the mmour.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • a compound of the invention Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable caniers.
  • the canier(s) must be "acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • any nucleic acid used in such methods is a nucleic acid capable of selectively hybridising to the gene conesponding to the cDNA insert of clone IMAGE 264611.
  • tumour suppressor gene that the gene is the gene conesponding to the cDNA insert of clone IMAGE 264611 or a suitable variant thereof, for example a truncated version or an intron-free version
  • SUBSTTTUTE SHEET such as a cDNA. It is particularly prefened that the polypeptide capable of being encoded by a nucleic acid comprising a tumour gene which nucleic acid is capable of selectively hybridising to the said region of human chromosome 10 is a polypeptide capable of being encoded by the gene conesponding to the cDNA insert of clone IMAGE 264611.
  • SSCP single-strand conformation polymo ⁇ hism
  • PCR polymerase chain reaction
  • YAC yeast artificial chromosome
  • CEPH Centre d'Etude du Polymo ⁇ hisme Humain.
  • Figure 1 shows a. Examples of allele loss at microsatellite markers on 10q23-q25 in prostate tumours. The upper boxed figure beneath each peak gives the length of the allelic fragment; the lower figure is the relative peak height. 'Shoulder' peaks to the left of the main peaks are due to polymerase slippage during PCR. b. Microsatellite instability. Instability, thought to result from DNA mismatch repair enors (10), was seen in 1/37 tumours at 21/24 loci. Fragment lengths are given beneath each peak. The example shown here probably reflects deletion of the 207bp allele in conjunction with expansion of the 213bp allele.
  • FIG. 2 shows allele loss at 10q23-q25.
  • Tumour numbers conespond to those in Figure 4.
  • Marker numbers in italics are D10S numbers (7).
  • Markers denoted 'AFM' have yet to be assigned D numbers; the full marker names are AFMa051tb9, AFMal24wd9, AFMa064za5, AFMa301exl and AFMa273yel.
  • Tumours 8, 16, 24, 30 and 31 also show allele loss at markers D10S189 and/or D10S570 on the p-arm of chromosome 10, implying whole chromosome loss. The smaller numbers give the approximate genetic distance between markers in centiMorgans.
  • SUBSTTTUTE SHEET (RULE 26) There is a clearly defined common region of deletion between markers AFMal24wd9 and D10S583, a distance of approximately 9 centiMorgans. By contrast, only tumours 1 and 11 show specific loss of markers around Mxil and in both instances this is in conjunction with allele loss in the AFMal24wd9- 7. S5S3 region.
  • Figure 3 shows Mxil loss in prostate tumours: assessment of allele loss at the (AAAAC) ⁇ polymo ⁇ hism in the 3' untranslated region of the Mxil gene in tumours 1 and 11 , which show specific loss of adjacent microsatellite markers, by fluorescence based typing.
  • the boxed numbers beneath each peak give the allele fragment length (upper) and relative peak height (lower).
  • Tumour 1 shows clear loss of Mxil (peak height reduction 58%) whereas tumour 11 shows no apparent loss of Mxil, despite showing loss of adjacent microsatellite marker AFMa273yel .
  • Table 2 shows the results of assessment of prostate tumours for 10q23-q25 loss.
  • Figure 4(a) is a physical map of the minimal region showing the position of the YAC clones and markers D10S541 and AFM337
  • Figure 4(b) is a more detailed map showing the position of BAC and PAC clones.
  • Figure 5 shows further, more informative, LOH data.
  • Tables 3 to 22 describe the sequenced inserts of the expressed sequence tags (ESTs) which are derived from the gene corresponding to the cDNA insert of IMAGE clone 264611.
  • Figure 6 shows the complete sequence of a cDNA of a particularly preferred nucleic acid molecule. Potential position of introns
  • SUBSTTTUTE SHEET (RULE 26) is shown (the "ss" above a dinucleotide denotes the splice site). The 3' untranslated sequence is in lower case.
  • Figure 7 shows the translation in one reading frame of the nucleotide sequence of Figure 6.
  • Example 1 Localization of a prostate tumour suppressor gene to the 10q23- ⁇ 24 boundary
  • Tumours and venous blood samples were obtained from men undergoing transurethral resection of the prostate. Tumour tissue was microdissected away from normal tissue and mmour and blood DNA prepared as described previously (6).
  • PCR was performed in 50 ⁇ l reactions containing 30ng template DNA, lx PCR buffer (Boehringer Mannheim), 20pmol primer, 20 ⁇ M dNTPs (Boehringer Mannheim) and 1 unit of Taq polymerase (Boehringer Mannheim) on a GeneAmp 9600 thermal cycler (Perkin-Elmer Cetus).
  • lx PCR buffer Boehringer Mannheim
  • 20pmol primer 20 ⁇ M dNTPs
  • Taq polymerase Boehringer Mannheim
  • GeneAmp 9600 thermal cycler Perkin-Elmer Cetus
  • one of the primers was tagged with a fluorescent label (JOE, FAM, HEX or TAMRA; Applied Biosystems).
  • Microsatellite reaction mixmres were given 30 cycles of 30 seconds @ 94° , 30 seconds @ 55° and 30 seconds @ 72° , preceded by a 3 minute hot start at 95° .
  • the annealing temperamre was lowered to 50° for amplification of Mxil helix-loop-helix and leucine zipper exons (5), and increased to 60° for amplification of the 3' exon; primer sequences for 3' exon amplification are 5'- GAGATTGAAGTGGATGTTGAAAG-3' (SEQ ID No 7) (A) and 5'- AAATACAGGTCCTCTGACCC-3' (SEQ ID No 8) (B) and give a 319 or 324bp product.
  • primer A was tagged with FAM.
  • Microsatellite allele sizes and loss of heterozygosity were determined by size separation of PCR products in a 6% denaturing polyacrylamide gel in the presence of a 2500-ROX size standard (Applied Biosystems) and detection with an 373A DNA sequencer running Genescan software (Applied Biosystems), following the manufacturer's guidelines. Up to 10 markers, distinguishable by size or fluorescent tag, were typed simultaneously. The resulting data were analysed using Genotyper software (Applied Biosystems).
  • PCR-amplified Mxil exons were sequenced using a PRISM cycle sequencing kit (Applied Biosystems) and a 373A DNA sequencer running 373A collection and analysis software (Applied Biosystems) in accordance with the manufacturer's instructions. Each exon was sequenced twice (once from each end) from independent PCR reactions. Sequence electropherograms were aligned using Sequence Navigator software (Applied Biosystems) and compared by eye.
  • the retinol binding protein 4 gene (RBP4) and the cytochrome P450IIC gene cluster (CYP2C) were positioned on the deletion map following the identification of yeast artificial chromosome (YAC) clones bearing both these loci and adjacent microsatellite markers D10S185 and D10S571 (11).
  • the map clearly reveals a common region of deletion proximal to RBP4 and CYP2C which have been cytogenetically mapped to 10q23-24 and 10q24.1 respectively (12, 13) ( Figure 2). This region is lost in all of the tumours showing lOq loss in our smdy, with the exception of mmour 37, which was not informative for the markers from this area.
  • Tumours 1 , 3, 6, 13, 14 and 15 define a minimal region of deletion between markers AFMal24wd9 and D10S583, a distance of approximately 9 centiMorgans.
  • SUBSTTTUTE SHEET (RULE 26) microsatellite marker D10S597 (14). Only two mmours, 1 and 11 , showed specific loss of markers immediately flanking Mxil and in both cases this was in conjunction with allele loss in the AFMal24wd9- D10S583 region ( Figure 2).
  • tumours 1 and 11 we screened tumours 1 and 11 , and those mmours showing loss of the entire region, for Mxil mutations by PCR amplification of individual exons followed by SSCP analysis (8).
  • Primers for PCR amplification of exons encoding helix-loop-helix and leucine zipper domains were taken from Eagle et al (5).
  • primers derived from the immediate 5' end of the exon and from within the 3' untranslated sequence were used (4, 5). These 3 pairs of primers give 66 % coverage of the coding sequence of Mxil .
  • the data presented here indicate the presence of a prostate tumour suppressor gene (or genes) at the 10q23-q24 boundary, and more specifically between markers AFMal24wd9 and D10S583, a region spanning approximately 9 centiMorgans. This region was deleted in 22 of 23 prostate mmours showing lOq losses, with the 23rd being uninformative for the relevant markers. lOq loss may be an early event in some instances of prostate carcinogenesis; losses were observed in early
  • Mxil has been shown to be mutated in prostate mmours, only a small proportion of cells in each mmour were found to be carrying Mx/7 mutations (5).
  • the authors offer two possible explanations. The first is that the mmours smdied may have contained significant amounts of non- neoplastic tissue. The second is that mutated Mxil alleles are only present in a small number of neoplastic cells. Given that we were unable to detect Mxil mutations in microdissected mmours containing ⁇ 30% contaminating normal tissue and showing a degree of lOq loss ranging from 25-79% (as estimated by microsatellite allele loss - see Table 2) the latter seems more likely.
  • Figures 4 and 5 give more detailed mapping data between AFM 124 and D10S583, the markers that define the minimal region in the manuscript, allowing us to nanow the ⁇ iinimal region further to the interval between D10S541 and D10S215; more particularly between D10S541 and AFM337xf9, a distance of less than lcM.
  • the physical mapping data are summarised below:
  • SUBSTTTUTE SHEET (RULE 26) from the CEPH mega- YAC library.
  • 7B-F12 and 24G-A10 are publicly available from the ICI YAC library. Both of these libraries are publicly available from the Human Genome Mapping Project Resource Centre, Hinxton Hall, Hinxton, Cambridgeshire, CBIO IRQ, UK. Sizes for mega- YAC clones are taken from CEPH data. ICI YAC clones were sized by us.
  • YACs 821-D-2, 831-E-5, 796-D-5, 24G-A-10 and 734-B-4 have been mapped in more detail to give a large scale restriction map of the region (see Figure 4). This contig does not include all restriction sites.
  • YACs 821-D-2 and 831-E-5 appear to be identical and span the minimal region (D10S541 - AFM337xf9). They therefore contain all or part of the mmour suppressor gene.
  • ESTs (Expressed Sequence Tags) are generated and assigned to genomic regions using the following procedure.
  • the polypeptide encoded by the cDNA insert of IMAGE clone 264611 has some similarity to the protein tensin and to auxilin, a protein involved in protein transport to the cell membrane via clathrin coated vesicles.
  • the gene conesponding to the cDNA insert of clone IMAGE 264611 is a mmour suppressor gene.
  • the prostate mmour suppressor gene or genes are identified by screening a panel of RNAs from prostate and other mmour cell lines, in order to identify an altered, usually reduced, level of transcript.
  • the transcript is likely to be large, as it will probably have a complex function and several sites for disabling mutation 'hits' (cf BRCA1 , RB).
  • Cross-species conservation is a good indicator that the gene has a basic cell 'housekeeping' function, the loss of which can lead to a loss of growth control and mmour formation.
  • the prostate mmour suppressor gene cDNA is identified as follows.
  • Part of one of the YAC clones is used as a probe to screen a prostate cDNA library directly following radiolabelling.
  • the 400kb Mlul fragment (marked on the restriction map in Figure 4), which covers about 75% of the minimal region, is used as a probe - this fragment can be separated cleanly from a pulsed field gel following digestion.
  • the entire 24G-A10 YAC is used as a probe.
  • a standard colony/filter hybridization approach is used.
  • Suitable BAC or PAC clones may also be used.
  • Mutation analysis of the entire coding region in tumours shows that the gene is a prostate mmour suppressor gene. This is done by analysing each exon individually for mutations. Methods for mutation analysis used are single-stranded conformation polymo ⁇ hism (SSCP) analysis (or variations of this technique) and direct DNA sequencing.
  • SSCP single-stranded conformation polymo ⁇ hism
  • Genes located within the region are identified by screening of cDNA libraries with the probes obtained from the human nucleic acid sequence contained within the YACs, BACs and PACs or by exon trapping methods or by sequencing of the human nucleic acid sequence contained within the YACs, BACs and PACs, automated sequencing techniques make this routine, and use of computer programs, eg GRAIL II, that distinguish coding sequence.
  • the results are confirmed by RT-PCR of prostate RNA from prostate tissue or a cDNA library.
  • the prostate mmour suppressor gene or genes are found to be expressed in normal prostate tissue, mutation analysis of the entire coding region shows that expression of the gene(s) may be altered in prostate tumours compared to normal prostate, the product of the genes may be truncated at the protein level, the mRNA product may be truncated, or have altered splicing compared to normal which results in an abnormal protein, the resulting protein encoded by the altered gene may have abnormal properties or distribution within the tissue.
  • Chromosomal deletions in a specific region on chromosome 10 are detected using inte ⁇ hase fluorescent in situ hybridisation (FISH) on cells in inte ⁇ hase to check for loss.
  • FISH fluorescent in situ hybridisation
  • SUBSTTTUTE SHEET (RULE 26) across a slide and the cell membrane permeabilised. This allows the reagents for in situ hybridisation to enter the cells containing inte ⁇ hase chromosomes.
  • the BACs or PACs or other suitable probes specific for the region deleted are hybridised to the chromosomes after labelling the probes with a fluorescent dye.
  • a chromosome containing a region of deletion shows no signal; and chromosomes from a cell in which one chromosome 10 has suffered a deletion from this region will show only one signal and not two. Therefore, a method is provided that can detect lOq deletions in biopsies from patients. These are useful indicators of the staging of the grade of the mmour between benign and malignant hype ⁇ lasia and may indicate that a more aggressive treatment regime should be undertaken.
  • Suitable YAC clones for use as probes, include 821-D-2, 831-E-5, 796- D-5, 24G-A-10 and 734-B-4.
  • Any of the BAC or PAC clones derived from the region of interest may be used and include 60C5 and 46B12.
  • nucleic acid which is capable of selectively hybridising to the gene conesponding to die cDNA insert of clone IMAGE 264611.
  • the gene itself, or a suitably sized fragment thereof, is particularly suited as a probe.
  • the probe is ideally between lOkb and 1Mb, preferably between 60- 200kb.
  • FISH is described by Bentz et al (1994) Leukemia 8(9), 1447-1452.
  • the BAC or PAC clone (such as BAC clone 60C5) is used on nuclei
  • SUBSTTTUTE SHEET (RULE 26) isolated from prostate tissue.
  • the method for isolating nuclei from frozen tissue is as follows.
  • Slides may be stored at this point as follows (dehydrate through ethanol series (75%, 85 %, 95% for 2 minutes each; air dry; store slides at -20°C with dessicant; store residual nuclear suspension in PBS at -70°C (it can be thawed x2 without any effect on the quality of the subsequent hybridization)). (12) Before hybridization the DNA needs to be denatured. Place slide on hot plate at 73 °C with 70% formamide/2xSSC pH 7.0 under a coverslip for 2.5 minutes. (13) Dehydrate in ethanol series of icecold 70% , 95% and 100% for 3 minutes each and air dry.
  • the BAC or PAC clone (eg BAC clone 60C5) is used as a diagnostic probe. The whole clone is used to generate a labelled probe.
  • the two probes are labelled differently so that they may be distinguished.
  • the probes are to be labelled by nick translation with biotin or digoxygenin using a commercially available kit (eg Bionick kit, Life Technologies).
  • Step 2 mouse anti-Digoxygenin FITC, and Avidin-Texas Red.
  • Step 3 rabbit anti-mouse FITC, and anti-Avidin Biotin.
  • Step 4 anti-rabbit FITC, Avidin-Texas Red.
  • Counterstain: DAPI (0.15 ⁇ g/ml 5 ⁇ l of 30 ⁇ l/ml stock solution + 995 ⁇ l glycerol (Cytofluor).
  • the 60C5 probe produces two signals (spots) per cell. Two spots per cell are also seen for the chromosome 10 centromeric
  • the inte ⁇ hase FISH method can be used using genomic clones in the region.
  • the genomic DNA is about 60-200 kb.
  • normal tissue shows two dots
  • mmour tissue shows one or no dots, or alternatively fewer dots than the number of chromosome 10 copies present in any cell.
  • Centromeric repeat sequences are used to demonstrate the presence of chromosome 10 in a cell.
  • efficiency is typically between 85 and 95 % , ie 85-95 nucleic per 100 show two signals. Efficiency is dependent on both the probe and the experimental conditions but may be optimised empirically.
  • Affected tissue shows a significantly greater percentage of cells with only a single signal. The presence in the sample of contaminating, normal, cells will prevent this percentage from reaching 100%. It is therefore desirable to dissect out the area of the cells prior to these assays.
  • the methods and outcomes are: (i) Take tissue sample from patient, dissect out/purify affected area of tissue, and extract nuclei. (ii) Label probe with detectable tag. (iii) Contact probe with prepared sample under hybridising conditions, (iv) Remove, by washing, non- hybridised excess probe. (v) Visualise hybridised probe. Probe hybridised to a single locus is visualised as a signal (spot) by microscopy, (vi) In unaffected tissue, the majority of cells are found to show two signals, per cell. A minority of cells may show less than two spots, due
  • a monoclonal antibody directed at the mmour suppressor gene product is labelled with 125 I.
  • a sample of prostate tissue is prepared and proteins separated by SDS-polyacrylamide gel electrophoresis. The proteins are electroblotted onto a nitrocellulose membrane and the membrane incubated with the monoclonal antibody.
  • Presence of the mmour suppressor gene product is detected.
  • the absence of the product indicates an increased susceptibility to prostate cancer.
  • the mmour suppressor gene is introduced into a patient who is susceptible to prostate cancer using a suitable retroviral vector.
  • Example 6 Use of IMAGE clone 264611 (and primer or probes derived therefrom) in diagnosing prostate cancer
  • Clone 264611 (and primers or probes derived from it) are used for detection of altered mRNA levels by in situ hybridisation, Northern
  • S BSTTT analysis also detection of altered mRNA species profile
  • quantitative RT-PCR quantitative RT-PCR.
  • expression detection methods other than in situ hybridisation
  • substantially pure mmour tissue In situ hybridisation uses fixed tissue.
  • a positive result indicative of prostatic cancer is altered expression levels compared to prostate tissue which is not cancerous or an altered pattern of transcript expression compared to normal prostate tissue.
  • Samples suitable for analysis also include fresh prostate tissue, tissue collected by needle biopsy from prostate or from metastasis.
  • PCR primers derived from the cDNA insert of IMAGE clone 264611 are used for RT-PCR followed by mutation detection or protein truncation assays.
  • a result indicative of prostate cancer is the detection of coding mutations, or a truncated protein product.
  • the methods of this Example are useful in detecting the presence of prostate adenocarcinoma.
  • Primers derived from intronic sequences of the gene conesponding to IMAGE 264611 are used to amplify the gene exons, which are then examined for mutations by various methods (sequencing, SSCP or any form of mismatch detection) or used in protein truncation assays. Suitable samples include fresh prostate mmour tissue, prostate cells recovered from blood, urine or semen, and DNA recovered from paraffin blocks.
  • DGGE direct sequencing
  • mis-match cleavage cleavage
  • heterozygote analysis cleavage
  • Loss of heterozygosity studies using markers D10S541 , D10S1765 (AFM337xf9) and D10S215 are used to determine loss of the D10S541- D10S215 interval.
  • markers consist of blocks of tandem CA repeats flanked by unique DNA sequence and are commonly known as microsatellites.
  • the number of CA repeats shows variation between alleles (homologs on different chromosomes). This may be exploited to distinguish the two homologous chromosomal regions bearing these markers in a given tissue.
  • biopsied prostate DNA eg from urine or semen
  • microsatellite profiles with those of DNA extracted from blood or cheek cells (eg by means of a mouthwash)
  • loss of one homolog of the D10S541-D10S215 interval in prostate tissue can be assessed.
  • This method is particularly useful for distinguishing between neoplasia (loss of one homolog) and hype ⁇ lasia (no loss) of the prostate.
  • PCR primer sequences are:
  • D10S215 5'-TGGCATCATTCTGGGGA-3' (SEQ ID No 3) 5'-GCTTTACGTTTCTTCACATGGT-3' (SEQ ID No 4)
  • D10S1765 5'-ACACTTACATAGTGCTTTCTGCG-3' (SEQ ID No 5)
  • Double deletion of the gene may be detected by analogous methods.
  • This mutation was detected following PCR amplification of exon 4 (using the intronic primers described in the figure of exon 4) and subsequent sequencing of the PCR product using standard metiiods.
  • a Staging is based on digital rectal examination and bone scan (9).
  • M.Fatima Bonaldo This library was constructed from the same fetus as the fetal lung library, Soares fetal lung NbHL19W. "
  • ORGANISM Homo sapiens Eukaryotae; mitochondrial eukaryotes; Metazoa;
  • REFERENCE 1 bases 1 to 414) AUTHORS Hillier.L. , Clark,N., Dubuque,T. ,
  • M.Fatima Bonaldo This library was constructed from the same fetus as the fetal lung library, Soares fetal lung NbHL19W. "
  • /lab_host "DHl OB (ampicillin resistant)" mRNA complement( ⁇ 1. . >414)
  • AAATTCAGAC TTTTGTAATT TGTGTATGCT GATCTTCATC AAAAGGTTCA 101 TTCTCTGGAT CAGAGTCAGT GGTGTCAGAA TATCTATAAT GATCAGGTTC 151 ATTGTCACTA ACATCTGGTG TTACAGAAGT TGAACTGCTA
  • RI sites of a modified pT7T3 vector (Pharmacia).
  • the retinas were obtained from a 55 year old Caucasian and total cellular poly(A) + RNA was extracted 6 hrs after their removal.
  • the retina RNA was kindly provided by Roderick R. Mclnnes M.D. Ph.D. from the University of Toronto. Library constructed by Bento Soares and M.Fatima Bonaldo. "
  • HUMGS0007741 Human Gene Signature, 3'-directed cDNA sequence.
  • SOURCE One or more human adult tissue.
  • ORGANISM Homo sapiens Eukaryotae; mitochondrial eukaryotes; Metazoa;
  • double-stranded cDNA was size selected, ligated to Eco RI adapters (Pharmacia), digested with Not I and cloned into the Not I and Eco RI sites of a modified pT7T3 vector (Pharmacia).
  • the retinas were obtained from a 55 year old
  • Gnathostomata Osteicluhyes; Sarcopterygii; Choanata; Tetrapoda; Amniota;
  • REFERENCE 1 bases 1 to 427) AUTHORS Hillier,L. , Clark,N., Dubuque,T. , Elliston,K. , Hawkins, M., Holman,M. , Hultman,M., Kucaba,T. , Le,M.,
  • RI 1st strand cDNA was primed with a Not I - oligo(dT) primer
  • double-stranded cDNA was size selected, ligated to Eco RI adapters (Pharmacia), digested with Not I and cloned into the Not I and Eco RI sites of a modified pT7T3 vector (Pharmacia).
  • the retinas were obtained from a 55 year old
  • Gnathostomata Osteichthyes; Sarcopterygii; Choanata; Tetrapoda; Amniota;
  • REFERENCE 1 bases 1 to 117 AUTHORS Hillier,L., Clark,N., Dubuque,T., Elliston,K., Hawkins, M. , Holman,M. , Hultman,M., Kucaba,T., Le,M.,
  • TCTCCCCTTC TACTGCCTCN 51 AACACGGCGG CGGCGGCGGC
  • GGCACATCCA GGGACCCGGG CCGGTTTTAA 101 ACCTCCCGTC CGCCGCC
  • RNA from normal foreskin melanocytes (FS374) was kindly provided by Dr. Anthony P. Albino.
  • Amniota Mammalia; Theria; Eutheria; Archonta; Primates; Catarrhini; Hominidae;
  • 1st strand cDNA was primed with a Not I - oligo(dT) primer double-stranded cDNA was size selected, ligated to Eco RI adapters (Pharmacia), digested witn Not I and cloned into the Not I and Eco RI sites of a modified pT7T3 vector (Pharmacia).
  • RNA from normal foreskin melanocytes (FS374) was kindly provided by Dr. Anthony P. Albino.
  • Amniota Mammalia; Theria; Eutheria; Archonta; Primates: Cata ⁇ hini; Hominidae;
  • RNA from normal foreskin melanocytes (FS374) was kindly provided by Dr. Anthony P. Albino.
  • Amniota Mammalia; Theria; Eutheria; Archonta; Primates; Catarrhini; Hominidae;
  • Trevaskis,E. Waterston,R., Williamson,A., Wohldmann,P. and Wilson,R.
  • CTTAAAATTT 251 GGAGAAAAGT ATCGGTTGGC TTTGTCTTTA
  • RNA from normal foreskin melanocytes (FS374) was kindly provided by Dr. Antbony P. Albino. ORGANISM Homo sapiens
  • Amniota Mammalia; Theria; Eutheria; Archonta; Primates; Cata ⁇ hini; Hominidae;
  • Vertebrata Gnathostomata; Mammalia; Eutheria; Primates; Catarrhini; Hominidae;
  • AGTTTATTCA 51 AGTTTATTTT CATGGTGTTT TATCCCTCTT
  • CTATACTGCA 401 AATGCTATCG ATTTCTTGAT CACATAGGGC
  • Vertebrata Gnathostomata; Mammalia; Eutheria; Primates; Catarrhini; Hominidae;
  • REFERENCE 1 bases 1 to 224) AUTHORS Choi,S.S. , Yun,J.W., Choi,E.K., Cho,Y.G., Sung,Y.C. and Shin,H.-S. TITLE Construction of a gene expression profile of a human fetal liver by single-pass cDNA sequencing JOURNAL Unpublished
  • Average insert size 1.0 kb; Uni-ZAP XR Vector; 5' adaptor sequence: 5'-GAATTCGGCACGAG-3'; 3' adaptor sequence: 5'-CTCGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • Wilson RK Wilson RK
  • REFERENCE 1 (bases 1 to 451) AUTHORS Hillier,L. , Clark,N., Dubuque,T. ,
  • Seq primer mob.REGA+ET High quality sequence stop: 240. FEATURES
  • Soares and M.Fatima Bonaldo This library was constructed from the same fetus as the fetal heart library, Soares fetal heart NbHH19W. "
  • Inserts from retina cDNA library DNA were isolated, randomly primed, PCR amplified, size-selected, and cloned into lambda gtlO. Individual plaques were arrayed and used as templates for PCR amplification, and these PCR products were used for sequencing.
  • /clone_lib "Human retina cDNA randomly primed sublibrary”
  • /sex “mixed (males and females)”
  • /tissue type "retina”
  • /dev_stage "adult”
  • /lab_host "E. coli strain K802" mRNA
  • GNGNNNTTNC TACTCANGAT CATTTGGNGG TTAAAGTAAG TACTAGATAN 51 TCCTTGTCAT TATCTGCACG CTCTATACTG CAAATGCTAT CGATTTCTTG 101 ATCACATAGA CTTCCATTTT CTACTTTTNC TGAGGTTNCC
  • ACATTGGGTC CTTTTTTAGC 201 ATCTGGTNCT GTGNGTGGAA
  • CAAACATCAN 401 CTTGGGAAAC AACAGGGNCA CTGGTCTTTT
  • ORGANISM Homo sapiens Eukaryotae; mitochondrial eukaryotes; Metazoa; Chordata; Vertebrata; Eutheria; Primates; Catarrhini;
  • Hominidae; Homo. REFERENCE 1 bases 1 to 601) AUTHORS Hillier,L. , Clark.N., Dubuque,T. , Elliston,K. , Hawkins, M. , Holman,M., Hultman,M. , Kucaba,T.,
  • Wilson,R. TITLE The WashU-Merck EST Project JOURNAL Unpublished (1995) COMMENT Contact: Wilson RK WashU-Merck EST Project
  • Seq primer mob.REGA+ET High quality sequence stop: 463.
  • double-stranded cDNA was size selected, ligated to Eco RI adapters (Pharmacia), digested with Not I and cloned into the Not I and Eco
  • AAAGTGGAGA 301 CAGACTGATG TGTATACGTA GGAATTTTTT
  • ORGANISM Homo sapiens Eukaryotae; mitochondrial eukaryotes; Metazoa;
  • M.Fatima Bonaldo This library was constructed from the same fetus as the fetal lung library, Soares fetal lung NbHL19W. "
  • ORGANISM Homo sapiens Eukaryotae; mitochondrial eukaryotes; Metazoa;
  • REFERENCE 1 bases 1 to 429) AUTHORS Hillier,L., Clark,N., Dubuque,T. ,
  • M.Fatima Bonaldo This library was constructed from the same fetus as the fetal lung library, Soares fetal lung NbHL19W. "
  • /clone "347316”
  • /clone_lib "Soares fetal heart NbHH19W”
  • AAATTCAGAC TTTTGTAATT TGTGTATGCT GATCTTCATC
  • AAAAGGTTCA 101 TTCTCTGGAT CAGAGTCAGT GGTGTCAGAA TATCTATAAT GATCAGGTTC
  • AATACTGCAA ATGGCTATCC GATTTCCTGG ATCCACCATA GGNCTTCCNA 401 TTTCCAACTT TTCCCTGNGG TTCCCCCGG

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Abstract

Un procédé pour déterminer la prédisposition d'un patient au cancer consiste (i) à prélever sur celui-ci un échantillon contenant un acide nucléique; et (ii) à mettre en contact ledit acide nucléique avec un acide nucléique susceptible d'hybridation sélective avec la région du chromosome humain 10 délimitée par l'ADN défini par les marqueurs D10S541 et D10S215, à condition que cet acide nucléique ne soit pas l'un quelconque de certains chromosomes artificiels de levure, chromosomes artificiels bactériens, chromosomes artificiels dérivés de P1 ou marqueurs de séquences exprimées définis dans le présent fascicule. Ledit acide nucléique est de préférence un gène suppresseur de la tumeur de la prostate.
PCT/GB1996/002588 1995-10-23 1996-10-22 Diagnostic de predisposition au cancer et traitement approprie WO1997015686A1 (fr)

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KR1019980702948A KR19990067011A (ko) 1995-10-23 1996-10-22 암에 대한 감수성 진단 및 그 치료방법
JP9516392A JP2000500329A (ja) 1995-10-23 1996-10-22 癌罹病性の診断およびその治療
BR9611255-7A BR9611255A (pt) 1995-10-23 1996-10-22 ácido nucleico, processos para determinar a suscetibilidade de um paciente a câncer, para diagnosticar câncer em um paciente, para prever as perspectivas relativas do surgimento particular de um câncer em um paciente, para tratar câncer e para determinar a perda de heterozigosidade em amostra de tecido, sistema para detectar a presença ou ausência de, ou mutação, na região de cromossomo 10 humano, polipeptídeo, molécula e uso de ácido nucleico e de uma molécula.
AU73161/96A AU722266B2 (en) 1995-10-23 1996-10-22 Diagnosis of susceptibility to cancer and treatment thereof
NZ320425A NZ320425A (en) 1995-10-23 1996-10-22 Diagnosis of susceptibility to cancer using nucleic acid probes
EP96935062A EP0859860A1 (fr) 1995-10-23 1996-10-22 Diagnostic de predisposition au cancer et traitement approprie
US08/906,156 US6287854B1 (en) 1996-10-22 1997-08-05 Diagnosis of susceptibility to cancer and treatment thereof
NO981662A NO981662L (no) 1995-10-23 1998-04-14 Diagnosering av disponerhet for kreft og behandling av dette

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WO1998033907A1 (fr) * 1997-01-30 1998-08-06 Board Of Regents, The University Of Texas System Suppresseur tumoral ts10q23.3
WO1999002704A3 (fr) * 1997-07-08 1999-04-01 Cold Spring Harbor Lab Phosphatase a double specificite et procedes d'utilisation
WO1999058665A3 (fr) * 1998-05-14 2000-04-20 Univ Virginia Isolement et utilisation de sequences exprimees dans le sinus urogenital d'un foetus
WO2000024933A1 (fr) * 1998-10-26 2000-05-04 John Wayne Cancer Institute Detection de la perte d'heterozygosite dans les tumeurs et le serum de patients atteints de melanome
US6287854B1 (en) 1996-10-22 2001-09-11 Imperial Cancer Research Technology Limited Diagnosis of susceptibility to cancer and treatment thereof
WO2002000929A1 (fr) * 2000-06-27 2002-01-03 Urogene Procede de diagnostic in vitro du cancer de la prostate et kit de mise en oeuvre
US6482795B1 (en) 1997-01-30 2002-11-19 Myriad Genetics, Inc. Tumor suppressor designated TS10q23.3
US6841350B2 (en) 1999-02-20 2005-01-11 The North West London Hospitals Nhs Trust Of Northwick Park Hospital Methods of diagnosing prostate cancer through the detection of the presence or absence of Pax 2 mRNA

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CN119061128B (zh) * 2024-11-06 2025-04-18 海南大学三亚南繁研究院 一种微卫星dna扩增引物对及二代测序方法

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6287854B1 (en) 1996-10-22 2001-09-11 Imperial Cancer Research Technology Limited Diagnosis of susceptibility to cancer and treatment thereof
US7217795B2 (en) 1997-01-30 2007-05-15 Myriad Genetics, Inc. Tumor suppressor designated TS10q23.3
US6262242B1 (en) 1997-01-30 2001-07-17 Board Of Regents, The University Of Texas System Tumor suppressor designated TS10Q23.3
US8735066B2 (en) 1997-01-30 2014-05-27 Board Of Regents, The University Of Texas System Tumor suppressor designated TS10Q23.3
US6482795B1 (en) 1997-01-30 2002-11-19 Myriad Genetics, Inc. Tumor suppressor designated TS10q23.3
WO1998033907A1 (fr) * 1997-01-30 1998-08-06 Board Of Regents, The University Of Texas System Suppresseur tumoral ts10q23.3
US7129040B2 (en) 1997-01-30 2006-10-31 Peter Steck Tumor suppressor designated TS10q23.3
WO1999002704A3 (fr) * 1997-07-08 1999-04-01 Cold Spring Harbor Lab Phosphatase a double specificite et procedes d'utilisation
WO1999058665A3 (fr) * 1998-05-14 2000-04-20 Univ Virginia Isolement et utilisation de sequences exprimees dans le sinus urogenital d'un foetus
WO2000024933A1 (fr) * 1998-10-26 2000-05-04 John Wayne Cancer Institute Detection de la perte d'heterozygosite dans les tumeurs et le serum de patients atteints de melanome
US6465177B1 (en) 1998-10-26 2002-10-15 John Wayne Cancer Institute Detection of loss of heterozygosity in tumor and serum of melanoma patients
US6841350B2 (en) 1999-02-20 2005-01-11 The North West London Hospitals Nhs Trust Of Northwick Park Hospital Methods of diagnosing prostate cancer through the detection of the presence or absence of Pax 2 mRNA
WO2002000929A1 (fr) * 2000-06-27 2002-01-03 Urogene Procede de diagnostic in vitro du cancer de la prostate et kit de mise en oeuvre

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JP2000500329A (ja) 2000-01-18
KR19990067011A (ko) 1999-08-16
AU722266B2 (en) 2000-07-27
EP0859860A1 (fr) 1998-08-26
NO981662D0 (no) 1998-04-14
AU7316196A (en) 1997-05-15

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