MATERIALS AND METHODS RELATING TO THE DIAGNOSIS AND PROPHYLACTIC AND THERAPEUTIC TREATMENT OF SYNOVIAL SARCOMA

Abstract: The invention provides a method for diagnosing the presence or absence of synovial sarcoma which comprises obtaining a sample derived from a tissue of a subject; contacting the sample with a specific binding member (sbm) which is specific for a binding partner (bp) which is characteristic of synovial sarcoma such that the presence of bp in the sample is diagnostic of synovial sarcoma, the bp being absent from an equivalent sample derived from a subject without synovial sarcoma; and detecting any binding of the sbm to its bp. Binding partners characteristic of synovial sarcoma are SSX and SYT sequences and fusion sequences thereof. Sequences, reagents and therapeutic methods and pharmaceuticals are also provided.

Description: Materials and Methods Relating to the Diagnosis and Prophylactic and Therapeutic Treatment of Synovial Sarcoma The present invention concerns materials and methods relating to the diagnosis and treatment (prophylactic and therapeutic) of synovial sarcomas. In particular, the present invention relates to diagnostic, prophylactic and therapeutic materials and methods based upon polynucleotides and polypeptides which are characteristic of synovial sarcoma and specific binding members therefor. In particular, the present invention relates to diagnostic materials and methods relating to the use of amplification techniques such as the polymerase chain reaction (PCR) to identify certain polynucleotide sequences characteristic of synovial sarcoma. Synovial sarcomas, which account for approximately 10% of soft tissue tumours, occur most commonly in young adults and are found at the extremities in the vicinity of the large joints 1 . Both biphasic and monophasic patterns of tumour histology can be distinguished. In the biphasic pattern, both epithelial-like cells and spindle cells are observed, whilst monophasic tumours contain only spindle cells. Cytogenetic studies have shown that approximately 70% of both biphasic and monophasic synovial sarcomas contain a characteristic chromosomal translocation t (X;18) (pll.2;qll.2) 2 - 8 . The presence of this translocation as the sole cytogenetic abnormality in some primary tumours indicates that its formation is a key molecular event in synovial sarcoma development . Somatic cell hybrids containing the derivative X chromosome that is formed by the t(X;18) translocation have been used to map markers and representative genes relative to the breakpoint. These studies allowed the applicants to delimit the breakpoint as follows: Xpter- DXS228- ( UBE1 - OATL1 - TIMP-OXS226 ) - (DXS255- TFE3 -ELK1 - DXS146) -OATL2-X;18- (DXS14-DXS422-DXS423-DXS674-DXS679) - Xcen 9"11 . A number of candidate genes from this region including ELK1 , TFE3 , TIMP, UBE1 , ARAF1 and SYN, which map to Xpll.2-p22.3, have also been assessed for involvement in the translocation, but no evidence of abnormal transcripts or rearrangement of these genes was found 6 . When fluorescence in si tu hybridization (FISH) was used to further localise the breakpoint, the applicants found that a 450kb Yeast Artificial Chromosome (YAC) , containing an ornithine-δ-aminotransferase region designated 0ATL2 (YAC OATL2.7) spans the position of the breakpoint 11 . The diagnosis of monophasic and biphasic synovial sarcoma is currently based upon histological appearance and immunohistochemical staining. Nonetheless it is still often difficult to distinguish between monophasic tumours and other spindle cell tumours such as leiomyosarcomas, some malignant fibrous histiocytomas, haemangiopericyt- omas, malignant peripheral nerve sheath tumours and fibrosarcomas 26 . Since treatment of soft tissue sarcomas depends on their type, it is important to have an accurate means of diagnosis. As a way to providing an accurate means of diagnosis, the applicants have used the YAC referred to above and repeat-free subclones derived from it, in strategies to isolate cDNA clones corresponding to the genes involved in the t(X,*18) translocation which is specific for synovial sarcomas. The present invention provides materials and methods both for establishing the presence or absence of characteristics specifically associated with synovial sarcoma (eg the t(X;18) translocation) as a way of diagnosing synovial sarcoma and for the prophylactic and therapeutic treatment of synovial sarcoma. Chromosome X comprises two breakpoints within two respective ornithine-δ-aminotransferase regions designated OATL1 and OATL2. Both OAT regions locate within the pll.2 region of chromosome X, with OATL2 locating closer to the centromere. Within the OATL1 region there is a nucleotide sequence region which the applicants have identified, characterized and designated SSX1 (standing for synovial sarcoma X chromosome break point 1) . Similarly, within the OATL2 region, there is a nucleotide sequence region which the applicants have identified, characterized and designated SSX2 (standing for synovial sarcoma X chromosome break point 2) . Chromosome 18 has a breakpoint in the qll.2 region within the nucleotide sequence for a gene newly identified and characterized by the applicants and which they have called the SYT gene (standing for synovial sarcoma translocation) . In translocation, there are breakages in the pair of chromosomes involved at their respective breakpoints. Where the breakage in the X chromosome is in the OATL1 region, part of the original SSX1 region is substituted with part of the SYT gene. Similarly, where the breakage in the X chromosome is in SSX2 region, part of the original OATL2 region is substituted with part of the SYT gene. The translocations have the effect of creating a new polynucleotide sequence which codes for a new fusion polypeptide/protein (which is coded for jointly by the two unrelated sequences brought together as a consequence of the translocation) which is absent from normal individuals in whom the translocation event has not occurred. PCR analysis demonstrates the presence of SYT- SSX1 or SYT-SSX2 fusion transcripts in 29 of 32 synovial sarcomas examined, demonstrating detection of transcripts to be a very useful diagnostic method. Furthermore the normal pattern for expression of SSXl, SSX2 and SYT nucleic acid and polypeptide sequences appears to be tissue specific. Thus in a normal individual, SSX sequences are only expressed at high levels in the testis. This means that one can also make a diagnosis by detecting the presence or absence of normal SSXl, SSX2 and/or SYT sequences (nucleic acid and/or polypeptide) in sites where the sequences are not substantially found in healthy individuals . The specific description relates the use of SYT probes to detect rearrangements of the SYT gene by eg Southern analysis. Thus the present invention provides the diagnosis of synovial sarcoma by use of specific binding members such as (a) nucleic acids hybridizable with a nucleic acid comprising either an SYT, SSXl or SSX2 polynucleotide sequence originating from a body site which does not normally show substantial expression of SYT or SSXl or SSX2 polynucleotide sequences; (b) a nucleic acid which hybridizes to both SYT and SSX (SSXl or SSX2) originating portions of a new polynucleotide sequence resulting from a translocation event; (c) substances comprising an antibody binding domain with specificity for either (i) one or more epitopes or sequences characteristic of either the SYT, SSXl or SSX2 polypeptide or (ii) one or more epitopes or sequences characteristic of a new polypeptide sequence of a fusion protein and not of the SYT, SSXl or SSX2 polypeptides. Where the antibody binding domain has a specificity for one or more epitopes characteristic of either the SYT, SSXl or SSX2 polypeptide it may be used to identify the presence or absence of such a polypeptide in a body site which does not normally substantially express that particular polypeptide. Thus normally, SSX polypeptides are expressed at high levels in the testis. The expression of SSX in body sites other than the testis such as the extremities of large joints, would be indicative of synovial sarcoma. In the above, reference is made to body sites which do not normally express the particular sequence (nucleic acid or polypeptide) (eg SSX) . These body sites include not only normal tissues, but also abnormal tissues such as tumours which do not express the particular sequence. Thus the presence of an SYT, SSXl or SSX2 sequence although characteristic of one tumour eg synovial sarcoma, may not be characteristic of another. Hence establishing the presence or absence of an SYT, SSXl or SSX2 sequence which is associated with synovial sarcoma, may allow one to distinguish between different sorts of tumours eg to distinguish a synovial sarcoma from another sort of tumour. This is important, as the treatment method of choice is often dependent upon the particular type of tumour a patient has. Where the specific binding member comprises nucleic acid, the member may simply be used as a specific probe in accordance with standard techniques and procedures. Alternatively, the specific binding member may comprise a pair of oligo- or polynucleotide sequences for use in an amplification technique such as PCR. Further if a new fusion protein resulting from a translocation event is detected as being "foreign" (ie other than native) by the body, there is the likelihood that the body will mount an immune response against it, generating antibodies with specificity for one or more epitopes of the new fusion protein. Similarly, where an SYT or SSX sequence is expressed at an abnormal body site the body may also mount an immune response, such that tnere may exist antibodies with specificity for one or more epitopes characteristic of the SYT or SSX polypeptide. Thus the present invention provides the diagnosis of synovial sarcoma by use of a specific binding member comprising one or more epitopes or sequences characteristic of either the SYT, SSXl or SSX2 polypeptide or of a new polypeptide sequence of a fusion protein and not of the SYT, SSXl or SSX2 polypeptides, in a specific binding assay to detect the presence or absence of such antibodies in a suitable sample obtained from an individual . The term specific binding pair is used to describe a pair of molecules comprising a specific binding member (sbm) and a binding partner (bp) therefor which have particular specificity for each other and which in normal conditions bind to each other in preference to binding to other molecules . Examples of specific binding pairs are antigens and antibodies, hormones and receptors and complementary nucleotide sequences. The skilled person will be able to think of many other examples and they do not need to be listed here. Further, the term "specific binding pair" is also applicable where either or both of the specific binding member and binding partner comprise just the binding part of a larger molecule. Thus in the context of antibodies, a specific binding member may comprise just a domain of an antibody (antibody binding domain) which is able to bind to either an epitope of an antigen or a short sequence which although unique to or characteristic of an antigen, is unable to stimulate an antibody response except when conjugated to a carrier protein. The present invention provides a binding member which is either: (i) specific for a new polynucleotide sequence resulting from a translocation event which is characteristic of synovial sarcoma (preferably where the specific binding member is for only part of the new polynucleotide sequence, it should be specific for a part which is characteristic of the new sequence and not of SYT, SSXl or SSX2) ; (ii) specific for a new polypeptide sequence of a fusion protein resulting from a translocation event which is characteristic of synovial sarcoma (preferably where the specific binding member is for only part of the new polypeptide, it should be specific for a part which is characteristic of the new polypeptide and not of the constitutive polypeptides SYT, SSXl or SSX2) ; (iii) specific for an antibody for either a new fusion protein produced by the patient as a result of a translocation event which is characteristic of synovial sarcoma or an SYT or SSX protein as a result of their formation in an abnormal body site; (iv) specific for either a SYT, SSXl or SSX2 polynucleotide sequence; and (vi) specific for a polypeptide coded for by either a SYT, SSXl or SSX2 polynucleotide sequences. In particular, the present invention provides oligonucleotide primer pairs for amplification of polynucleotide sequences (be they in the form of DNA, RNA, single-stranded or double-stranded) which comprise part or all of a SYT, SSXl and/or SSX2 polynucleotide sequence, or a polynucleotide sequence spanning the breakpoint (which spanning polynucleotide sequence may comprise part or all of the new polynucleotide sequence created by the translocation event) . In which case, the oligonucleotide primer pairs are designed to hybridize target regions of the new polynucleotide sequence, which are spaced apart from one another with the breakpoint locating between them. The primer pairs may be designed by use of the sequence information herein provided. Having increased the copy number of an SYT, SSXl or SSX2 polynucleotide sequence, or of a new polynucleotide sequence (or part of it spanning the breakpoint) , the amplified sequences may be readily detected by standard methods such as the provision of radioactive nucleotides for inclusion in the sequences being copied, ethidium bromide staining, sequencing and hybridization probing. Of course, no amplification would occur if polynucleotide sequence comprising targets for the primers is absent from the sample selected for testing. Thus where the sample is taken from a tissue eg a tumour around the extremity of a large joint, amplification of eg the SSX polynucleotide sequence (DNA or R A) would be indicative of synovial sarcoma, as SSX sequences (polynucleotide or polypeptide) are not normally present at this site. In contrast, no amplification would be indicative of the absence of synovial sarcoma. Similarly where primers designed to amplify a sequence portion which is characteristic of a new polynucleotide sequence created by a translocation event are used (eg a portion of sequence around the breakpoint) , any amplification would be indicative of synovial sarcoma, whereas no amplification would be indicative of the absence of synovial sarcoma. The present invention also provides a method for diagnosing synovial sarcoma by taking a suitable sample from a patient, and detecting the presence or absence of a new polynucleotide sequence resulting from a translocation event which is characteristic of synovial sarcoma by adding to the sample suitable oligonucleotide primer pairs (see above) , and other standard ingredients for carrying out a polynucleotide sequence amplification (an amplification based on a DNA template or an RNA template) , and applying standard hybridization, elongation and denaturation or strand separation conditions to amplify any new polynucleotide sequence positioned between the two primers and looking for the presence or absence of an amplification product or products, to determine the presence or absence of the t(X,*18) translocation. Also provided is a method for diagnosing synovial sarcoma by taking a sample from a patient from a site which does not normally have high levels of expression of either the SYT, SSXl or SSX2 polynucleotide sequence, and detecting the presence or absence of one or more of these polynucleotide sequences in that sample by adding to the sample suitable oligonucleotide primer pairs as above, and other standard ingredients for carrying out a polynucleotide sequence amplification (an amplification based on a DNA template or an RNA template) , and applying standard hybridization, elongation and denaturation or strand separation conditions to amplify any SYT, SSXl and/or SSX2 polynucleotide sequences present in the sample and looking for the presence or absence of an amplification product or products, in the sample. The primers may be designed from any part of the polynucleotide sequences for SYT, SSXl and SSX2 or from any part of a new polynucleotide sequence created by a translocation event (eg as now provided by the applicants) . It may be helpful, but not essential, to know the distance between the two primers, as this will aid in the analysis of the amplification product. The length of the primers should be such that they efficiently hybridize with good specificity. Typically the primers may be upwards of about 14 nucleotides. Generally, the primers may be 18-20 nucleotides. It is not necessary to have 100% correspondence between the primers and their target region in the SYT, SSXl or SSX2 polynucleotide sequence or in the new polynucleotide sequence. The primers may comprise one or more non- complementary bases. All that is necessary is that the primer and target sufficiently correspond for specific hybridization to allow the desired amplification reaction to proceed. The present invention also provides nucleic acid which comprises an oligo- or polynucleotide sequence hybridizable to either: (i) a polynucleotide sequence which codes for part or all of either the SYT amino acid sequence shown in Fig. 4a or the SSXl or the SSX2 amino acid shown in Fig. 7; or (ii) a polynucleotide sequence which codes for part or all of a polypeptide having part or all of the amino acid sequence shown in Fig. 4b; or (iii) a polynucleotide sequence which codes for part or all of a polypeptide having part or all of the amino acid sequence shown in Fig. 5. Generally speaking it is intended that alleles and derivatives (by deletion, insertion, substitution or inversion) of the polynucleotide sequences given in the Figures are included within the scope of the present invention, in that such alleles or derivatives will code for at least part of the given amino acid sequences. The present invention also provides a nucleic acid which comprises an oligo- or polynucleotide which comprises part or all of a polynucleotide sequence as shown in either Fig. 4a, Fig. 4b or Fig. 5 or Fig. 7 or a sequence complementary thereto. The present invention also provides a nucleic acid which comprises a polynucleotide having part or all of a polynucleotide sequence which comprises the SSX2 polynucleotide sequence of Fig. 4b or the SSXl polynucleotide sequence of Fig. 5. Thus the polynucleotide may comprise a part of the SSX2 polynucleotide, or a part of the SSXl polynucleotide not given by Figs . 4b and 5. The nucleic acid, oligo- or polynucleotide may be substantially free of other substances ie isolated and substantially pure. The present invention also provides recombinant transfer vectors and expression vectors which contain a nucleic acid or oligo- or polynucleotide as described above. The nucleotide sequences may be present in association with suitable control sequences such as promoters . The present invention also provides recombinant host cells which comprise such a transfer vector or expression vector. The recombinant host cells may be used to prepare polypeptides homologous to the polypeptides coded for by the SYT, SSXl or SSX2 polynucleotide sequences, or to part or all of a fusion protein coded for by a new polynucleotide sequence created by the translocation. Polypeptide homologues may be used in diagnostic tests to test patient samples for the presence or absence of antibodies with specificity for one or more epitopes characteristic of the SYT, SSXl or SSX2 proteins, or of the new fusion protein (see earlier discussion) . Polypeptide homologues may also be used to raise antibodies (monoclonal or polyclonal) with specificity for one or more sequences or epitopes characteristic of the native SYT, SSXl, SSX2 or fusion proteins. As mentioned earlier, polynucleotides as described above may be used as specific hybridization probes to determine the presence or absence in samples from patients of either the SYT, SSXl or SSX2 polynucleotide sequences, or of a new polynucleotide sequence resulting from a translocation. Where the probe is for a new polynucleotide sequence resulting from a translocation, preferably it may bind to a part of the new polynucleotide sequence which comprises nucleotides to either side of the breakpoint, in which case the probe would bind to sequences deriving from both SYT and SSXl or SSX2. Probes may be directed to DNA or mRNA complementary thereto. The probes maybe either cDNA probes, RNA probes or oligonucleotides. The probes may be suitably labelled in accordance with standard procedures to aid detection of hybridization. Commonly the labels maybe radio-, fluoro or enzyme- labels. Hybridization may be carried out in accordance with well known methodologies. The use of high stringency conditions will serve to minimise non-specific binding and the occurrence of false positives. The present invention also provides a polypeptide having (i) part or all of either the SYT amino acid sequence shown in Fig. 4a or the SSXl or the SSX2 amino acid sequence shown in Fig. 7; or (ii) part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4b; or (iii) part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 5. The polypeptides may be immunologically cross-reactive with a native SYT, SSXl, SSX2 or fusion protein. They may be immunologically cross-reactive with a polypeptide having an amino acid sequence as shown in either Fig. 4(a) , Fig 4(b) , Fig 5. or Fig. 7. The polypeptide may comprise a part of the SSX2 polypeptide or a part of the SSXl polypeptide not given by Figs . 4b and 5. The polypeptides may be substantially free of other substances ie isolated and substantially pure. Derivative polypeptides retaining the cross-reactivity are also provided. The polypeptides above may be prepared by recombinant methodologies or by methods of standard chemical synthesis. As stated above, the polypeptides may be used in diagnostic assays, or to raise antibodies. The present invention also provides specific binding members comprising an antibody binding domain with specificity for one or more epitopes characteristic of the SYT, SSXl or SSX2 proteins, or of the new fusion protein and not of SYT, SSXl or SSX2 proteins. The specific binding members may comprise antibodies, either monoclonal or polyclonal . Alternatively they may comprise derivatives, synthetic analogues or fragments of such antibodies which retain an antibody binding domain with the specificity described above. In practice, diagnostic methods utilising specific binding methods comprising an antibody binding domain (see above) may be particularly favoured (perhaps for reasons of simplicity, cost and effectiveness) . The applicants' provision of the sequences herein, allows one of ordinary skill in the art to make monoclonal and polyclonal antibodies which specificity for the desired polypeptide/protein by the utilisation of standard procedures well known in the art. Of course having once produced an antibody, they may be altered to produce antibody derivatives, fragments or functional equivalents (with respect to specificity) which whilst differing from the original antibody, retain an antibody binding domain of required specificity. The term "specific binding members comprising an antibody binding domain" as used herein hence covers both monoclonal and polyclonal antibodies as well as fragments, derivatives and functional equivalents thereof . The present invention also provides methods of diagnosing synovial sarcoma by detecting the presence or absence of the new fusion protein, or the new nucleotide sequence by use of a specific binding member as described above, or by detecting the presence or absence of SYT, SSXl or SSX2 proteins or polynucleotide sequences in samples from tissues which do not normally express these proteins. Diagnostic kits are also be provided for each of the above mentioned embodiments which comprise a specific binding member as described above, along with other reagents required to conduct the diagnostic test. The cytogenetically defined translocation t(X,*18) (pll .2;qll.2) found in human synovial sarcomas involves the joining of the chromosome 18SYT gene to either of two genes SSXl or SSX2 at Xpll 2. The fusion genes are then transcribed to produce SYT-SSX1 and SYT- SSX2 hybrid transcripts that are in turn translated to form SYT-SSX1 or SYT-SSX2 fusion proteins. Antisense oligonucleotides can be used against native SYT, SSXl or SSX2 sequences as a therapeutic treatment. Alternatively the SYT-SSX1 and SYT-SSX2 sequences or junctions can be targeted with antisense oligonucleotides. Thus antisense RNA technology can be applied to prevent or inhibit mRNA expression of a sequence characteristically associated with synovial sarcoma. Hence in a further aspect the invention provides a polynucleotide sequence that can be transcribed to produce RNA that is at least in part complementary to a SYT, SSXl, SSX2, SYT-SSXl or SYT-SSX2 mRNA and wherein the complementary portion of said transcribed RNA is of sufficient length to inhibit translation of said mRNA in order to inhibit production of a polypeptide encoded by an SYT, SSXl, SSX2, SYT-SSXl or SYT-SSX2 polynucleotide sequence. Suitably the complementary (or "antisense") RNA is homologous with, or complementary to, at least a portion of the nucleic acid shown in either Figure 4 (a) , Fig 4(b) , Fig. 5 or Fig. 7 or a variant or allele thereof and which is effective in blocking expression of DNA. Alternatively the effect of the proteins (SYT, SSXl, SSX2, SYT-SSXl or SYT-SSX2) can be blocked at the protein level . This may be achieved by administering a specific binding member comprising an antibody binding domain as earlier described. The specific binding member may be an antibody and suitably monoclonal. Antibodies can be raised in accordance with conventional methods known in the art (see earlier) . The use of the sequences provided herein and variants, derivatives or fragments thereof which are antigenically cross-reactive in the generation of antibodies forms a further aspect of the invention. The antibodies, where for use in therapy, may be "humanised" for example using methods known in the art, in order to reduce the possibility of harmful hypersensitivity reactions occurring in human patients. For example, the tail region of a non-human antibody with the desired specificity may be exchanged for that of a human antibody. A more complete humanisation may be effected by exchanging further framework regions as is known in the art. This may be done at the DNA level using recombinant techniques as known and used in the art. The invention further provides polynucleotide eg cDNA which encodes the above specific binding members with an antibody binding domain. The nucleic acid, oligo and polynucleotide sequences provided herein may also be inserted into suitable vectors for vaccine use eg pox virus vectors such as vaccinia. The vectors may also include appropriate control elements . Such vectors may be used for prophylactic or therapeutic vaccination in order to express in a controlled fusion SYT, SSX or SYT-SSX polypeptide sequences and hence stimulate an immune response thereagainst to prevent or mitigate any effects of sarcoma. Thus the invention relates to the therapeutic application of various agents including anti-sense RNA constructs, substances comprising an antibody binding domain and vaccine vectors in the treatment of synovial sarcoma. Preferably these agents will be administered in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier or diluent. Suitable agents may be combined, conjugated or complexed to a targeting agent, such as an antibody or antibody binding domain, which binds a synovial sarcoma antigen. Such antigens, targeting agents and means of combining them with the agents of the invention would be derivable by the skilled person. In some sarcomas (STS 159 and STS 253) an 87bp sequence that was shown by PCR and Southern hybridization to map to the X chromosome was found between previously identified SYT and SSX2 sequences. This insert sequence (polynucleotide or polypeptide) may also serve as a useful diagnostic marker for synovial sarcoma. Thus all the previously stated aspects of invention in relation to the SYT, SSXl and SSX2 may also be applied in relation to tne insert sequence eg the present invention provides the diagnosis of synovial sarcoma by use of specific binding members such as (a) nucleic acids hybridizable with a nucleic acid comprising an insert sequence as above (b) substances comprising an antibody binding domain with specificity for one or more epitopes or a sequence characteristic of an insert amino acid sequence. The above embodiments may be carried out using the information supplied herein along with standard techniques known in the art or with reference to such text books as Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989. In order that the present invention is more clearly understood, embodiments will now be described by way of example only and not by way of limitation with reference to the following examples in which: Fig. 1. (a) Restriction map of a 32kb region of the 450kb YAC OATL2.7. This 32kb region is present in cosmids designated e and 52 of C3 described in ref 12. The repeat-free region was identified by hybridization of restriction enzyme digested cosmid DNA to a probe prepared from human Cotl DNA. (b) Schematic representation of human SYT and synovial sarcoma SYT-SSX2 cDNA sequences. The positions of the SYT (filled) and SSX2 (cross-hatched) open reading frames are shown. The vertical arrow indicates the position of the t(X,*18) translocation. The position of the subfragments (I-V) used as hybridization probes are shown. The "*" indicates the position of the putative ligand that may bind to SH3 domains and "•" indicates the position of the putative ligands that may bind to SH2 domains. Restriction enzyme abbreviations: B, BamHl; Bg; Bglll; R; EcoRI; RV; EcoRV; and H, Hindlll. Fig. 2. Southern blot analysis. (a) Hybridization of probe V from the 3 ' end of the overlapping clones isolated from the A2243 cDNA library to the following BamHl or EcoRI digested DNAs: 255, SS255 synovial sarcoma cell line; A2243, A2243 synovial sarcoma cell line; LCL, LCL127 (GM01416) human cells containing four copies of the X chromosome; L2.7 and L2.10, YACs of 450kb and 300kb designated, respectively, 0ATL2.7 and OATL2.10 (ref 12) that span the 0ATL2 region; LI.2, a YAC of 650kb designated OATL1.2 (ref 12) that spans the OATL1 region,* 18, the DL18TS human-hamster somatic cell hybrid containing chromosome 18 as its only human component; H, hamster; and X, the C12D human-hamster somatic cell hybrid containing chromosome X as its only human component. (Jb) Hybridization of the same DNAs digested with PstI to probe III from the 5' end of the Group B cDNAs (Fig. lb) . N represents DNA from normal human lymphocytes. (c) Hybridization of BamHl digested DNA to a SYT gene probe (probe III) . DNA was from the SS255, HS-SY-II, SS217, SS223, SS153, SS125 and SS114 synovial sarcomas. For tumours SS125 and SS114 paired tumour (T) and blood (B) samples were examined. Fig. 3. (a) Expression of chromosome 18 SYT sequences (probe III, Fig. lb) and chromosome X SSX2 sequences (probe IV, Fig. l ) determined by Northern analysis of cytoplasmic RNA. RNA was from: 255 and 2243; the SS255 and A2243 synovial sarcoma cell lines; RB6 and RB15, the Rag/225/6 and Rag/255/15 human-mouse somatic cell hybrid lines containing the der(X) chromosome formed by the t(X;18) translocation; R, the Rag mouse cell line; and MH, the MNNG-HOS chemically-transformed human osteosarcoma cell line. The 3.7kb transcript SYT detected in synovial sarcomas and MNNG-HOS cells in this figure was also observed in A431 epidermoid carcinoma cells, A673 rhabdomyosarcomas, HTB86 Ewings sarcoma cell lines and at low levels in the SK-UT-1 leiomyosarcoma /02641 PC17GB95/01704 cell line. The 2.3-2.4kb SSX2 transcript was not detected in MNNG-HOS cells, A431 cells, A673 cells and HT1080 human fibrosarcoma cells. A 1.6kb SSX2 transcript was, however, detected in HT1080 cells (Jb) Detection of SYT-SSX2 hybrid transcripts by RT-PCR. PCR was performed using SYT (5' -CAACAGCAAGATGCATACCA-3 ' ) and SSX2 (5'- CACTTGCTATGCACCTGATG-3' ) primers to amplify reverse transcribed total RNA from synovial sarcomas (2243, 124, 498, 114, 159 and 125) and the following human tumour and leukaemia samples: CEM, ALL cell line; HTB86, Ewing sarcoma cell line; A431, epidermoid carcinoma cell line; 229 biphasic leukaemia; 82, neuroepithelioma; and 364, haemangioma. Control experiments using actin primers demonstrated that each sample of reverse transcribed RNA could yield PCR products. Fig. 4. (a) The human chromosome 18 SYT cDNA nucleotide sequence and predicted amino acid sequence (Accession Number X79201) . The open reading frame extends to the 5' end of the nucleotide sequence and the protein is numbered from the first methionine. The arrows show the position at which rearrangement occurs in synovial sarcomas. The eight C-terminal amino acids shown in bold type are removed by rearrangement with SSX2 sequences. (Jb) The human SYT-SSX2 cDNA nucleotide sequence and predicted amino acid sequence (Accession Number X79200) . The vertical arrow shows the position of the transition between SYT and SSX2 sequences . Amino acids encoded by the SSX sequences are shown in italics. The numbering of nucleotide and protein sequences is consistent with that used for the SYT cDNAs. The nucleotide sequence obtained for SYT-SSX2 cDNAs extends 5' to position 3 of the SYT cDNA. 5' to the breakpoint SYT and SYT-SSX2 cDNA sequences were identical. For both SYT and SYT-SSX2 protein sequences the position of the putative ligand that may bind to SH3 domains is underlined once and the putative ligands that may bind to SH2 domains are double underlined. Fig. 5. The human SYT-SSXl cDNA nucleotide sequence and predicted amino acid sequence. The vertical arrow shows the position of the transition between SYT and SSXl sequences . Fig. 6 Schematic representation of the SYT-SSXl protein junctions together with nucleic acid and protein sequences. (A) The SYT-SSXl junction originally identified in the HS-SY-II cell line (B) . The SYT-SSX2 junctions originally identified in the A2243 cell line. (C) The SYT-SSXl junction present in STS498. (D) Representation of a complex fusion that is found in tumours STS 159 and STS253. In these two tumours an 87bp sequence shown by PCR and Southern hybridization to map to the X chromosome was found between previously identified SYT and SSX2 sequences. Hatched boxes denote SYT protein and the filled boxes represent SSXl and SSX2 sequences. Numbers above the boxes indicate the amino acids at the position of the junction. The number of tumours which contain each type of junction are listed. Fig. 7 The human SSXl and SSX2 cDNA nucleotide and predicted amino acid sequences. The amino acid sequence is numbered from the methionine predicted to act as the site of translational initiation. The position of the SSXl and SSX2 breakpoint found in the majority of synovial sarcomas is indicated. The sequences 3' to this breakpoint which are present in the SYT-SSXl and SYT-SSX2 hybrid transcript are shown in bold type. A polymorphism (T→C) is found at position 634 in approx 50% of the sarcomas with translocations involving SSXl. The SSXl and SSX2 sequences can be distinguished by digestion with Smal and Lspl . Smal only digests SSX2 while Lspl only digests SSX2. The Smal and Lspl restriction sites are shown. Two methods have been used to select clones from a cDNA library that was prepared using RNA from the A2243 synovial sarcoma cell line. In the first approach the library was plated at low density and screened using a probe prepared from the 450kb YAC 0ATL2.7. Of the plaques detected by the OATL2.7 YAC, 25% also hybridized to an OAT cDNA probe indicating that they had been located by hybridization to the OAT pseudogene sequence present in the YAC. Based on the presence of common restriction maps and cross hybridization many of the remaining cDNA clones could be assigned to two groups. The applicants found that one group hybridized to an expressed sequence designated SB1 . 8 (DXS423) that had previously been cloned by an independent method (K. Davies, personal communication) . It was determined that these SB1.8 sequences map back to the YAC OATL2.7. The second group of cDNAs had a common 3' end and varied in length from 1.0-2.4kb. In the second approach the library was screened with single copy probes isolated from the OATL2 region. In previous studies cosmids corresponding to the OATL2 region 12 and mapped into 4 contigs of lOOkb (Cl) , 55kb (C2) , 80kb (C3) and 65kb (C4) were isolated. Hybridization of these cosmids to a human Cotl DNA probe identified a 14kb region that was free of high copy repeat sequences in contig C3. A restriction map of a 32kb region of contig C3, which contains this region is shown in Fig. la. Two clones from this repeat-free region (I and II, Fig. la) were used to screen the A2243 cDNA library. Restriction mapping of 4 clones identified in these studies revealed that 2 clones were members of the group of non-QAT, non-SB1 . 8 cDNAs isolated by screening with the YAC 0ATL2.7. To determine the chromosomal origin of the unidentified group of cDNAs, individual restriction fragments were hybridized to 0ATL2 YAC DNA and to DNA from somatic cell hybrids that contain individual human chromosomes. A probe from the 3' region (probe V, Fig. IJb) hybridized to multiple restriction fragments in BamHl and EcoRI digested human DNA (Fig. 2a) . The restriction fragments appeared to correspond to sequences present in YACs spanning the 0ATL2 regions and were present in a cell line (C12D) which contains the X chromosome as its only human component (Fig. 2a, lane X) . There was no hybridization to DNA from a human-hamster hybrid cell line containing chromosome 18 as its only human component (Fig. 2a, lane 18) . When taken together these observations demonstrate that this 3' probe detects sequences that map to the X chromosome. These 3' cDNA sequences mapping to chromosome X have been designated SSX2 (for synovial sarcoma X chromosome breakpoint) . Probe III from the 5' end of the cDNA hybridized to fragments of 10.5, 9, 5.5 and 3kb in PstI digested human DNA (Fig. 2Jb) as well as related sequences present in hamster DNA. The 9, 5.5 and 3kb fragments were retained in the human-hamster hybrid line containing chromosome 18 as its only human component (Fig. 2Jb, lane 18) but were not present either in the OATL pseudogene YACs or in the C12D cell line. These observations demonstrate that the sequences hybridising to probe III map to chromosome 18. To confirm this assignment probe III was used to isolate a human genomic DNA clone from a library prepared in the EMBL4 bacteriophage vector. When an isolated genomic clone containing a 15kb insert was used as a probe in FISH studies, hybridisation was observed at the qll.2 region of chromosome 18. The 5' cDNA sequence that maps to chromosome 18 has been designated SYT (for synovial sarcoma translocation) . A lOkb human PstI DNA fragment was also detected by probe III in human DNA but this fragment apparently did not map either to chromosome X or to chromosome 18. This sequence might represent a SYT- related gene or a SYT pseudogene sequence. A probe from the SYT gene (probe III, Fig. lb) hybridized to a large restriction fragment of ~50kb and to a smaller fragment of llkb in BamHl digested human DNA (Fig. 2c) . Notably, additional restriction fragments in the size range 17-23kb were detected in BamHl digested synovial sarcoma DNA. When blood-tumour pairs from the same patient were examined the additional fragment was detected in tumour but not in the corresponding blood (Fig. 2c, lanes 125 and 114) . An abnormal BamHl fragment was detected in 10/13 synovial sarcoma tumour samples examined. These studies demonstrate that probe III detects gene rearrangements of the SYT gene in approximately 75-80% of synovial sarcomas. The results obtained when Northern blots of tumour and cell line RNA were hybridized to chromosome X and chromosome 18 probes are shown in Fig. 4. The chromosome 18 SYT probe detected a 3.7kb transcript both in synovial sarcomas and in a variety of other human cell lines (Fig. 3a) . In addition a unique major 2.3-2.4kb transcript was detected in synovial sarcoma cell lines. This transcript was not detected in other human cell lines but was found in Rag/225/6 and Rag/255/15 somatic cell hybrid cell lines that contain the derivative X chromosome formed as a result of the t (X;18) (pll.2,*qll.2) translocation in the absence of other material from chromosomes X and 18 (Fig. 3a) . A transcript of 2.3-2.4kb was also detected in synovial sarcomas and in the Rag/255/6 and Rag/255/15 hybrids when an X chromosome SSX2 sequence was used as a hybridization probe (Fig. 3a) . In screening of lines from other types of human tumours we have only detected expression of SSX2 in one fibrosarcoma cell line (HT1080) which contained a 1.6kb SSX2 transcript (result not shown) . These observations indicate (i) that the normal SYT gene encodes a transcript of 3.7kb transcript that is expressed both in synovial sarcomas and in other human cell lines and (ii) that synovial sarcomas express a unique 2.3-2.4kb hybrid transcript that contains both SYT and SSX2 sequences. A SYT gene probe (probe III, Fig. IJb) was used to isolate cDNAs from a library prepared using RNA isolated from the M426 fibroblast cell line. Clones ranging in size from 2.2kb to 3.8 kb were isolated. Sequencing of these clones generated a continuous sequence of 3071bp that contained an open reading frame encoding a protein of 404 amino acids (Fig. 4a) . The SYT protein is rich in glutamine (19%) , proline (16%) and glycine (14%) . In database searches the SYT protein and nucleic acid sequences failed to exhibit homology to sequences in the EMBL, GeneBank and SWISS PROT databases except for the detection of weak homologies (20-25%) to regions of a variety of proteins that were also rich in glutamine and/or proline. The SYT protein contains no motifs suggestive of biochemical or functional properties such as putative membrane spanning helices, DNA binding domains or nucleotide binding sites with the exception of the presence of several regions that were candidate ligands for binding to SH3 and SH2 domains. The consensus sequence for ligands of SH3 domains is pφPpXP where P represents proline, p represents residues that are usually proline and φ represents a hydrophobic residue 13 . There is also a preference for proline at positions -5 and +4 relative to the central P. The SH3 binding regions are in general highly proline rich. This residue promotes the formation of a left-handed type II polyproline helix conformation that is adopted by ligands that bind to SH3 domains. Sequences in SYT that were examined centred around prolines at positions 93, 101, 112, 121, 269, 360 and 383 but the best match occurred with amino acids 378-387 (-5) PTQPGPPQPP (+4) which contains prolines at all of the positions mentioned above (Fig. 4a) . The SYT protein sequence also contains a consensus sequence for binding to the SH2 domain of phosphatidylinositol 3-kinase (YXXM) at position 67 and two consensus sequences for binding to the SH2 domains of Grb2 (YXNX) at positions 362 and 399 (ref. 14) . There are several consensus sites for glycosylation and phosphorylation, including one site for tyrosine phosphorylation at position 67. Sequencing of the hybrid SYT-SSX2 cDNAs indicates that the transcript produced by the rearranged SYT gene encodes a SYT-SSX2 fusion protein in which the C-terminal 8 amino acids of the SYT protein are replaced by 78 amino acids encoded by SSX2 sequences (Fig. 4Jb) . Notably, the C-terminal 8 amino acids of SYT that are removed during formation of SYT-SSX2 contain one of the consensus sequences for binding to the SH2 domain of Grb2. The SSX2 protein and nucleic acid sequences also failed to exhibit significant homology to sequences present in the EMBL, GeneBank and SWISS PROT databases. The SYT-SSX2 sequence extends to nucleotide 3 in the normal SYT cDNA sequence shown in Fig. 4a and an exact match was observed between SYT-SSX2 and normal SYT sequences 5' to the breakpoint. The open reading frame for SYT and SYT-SSX2 extends to the 5' end of the cDNA sequence. This may indicate that the SYT and SYT-SSX2 proteins are slightly larger than shown in Fig. 5 and that additional 5' sequences remain to be cloned. It is also possible that translational initiation may occur at methionines within the sequences shown in Fig. 4a. In analyses of an additional 36 SYT and SYT-SSX2 cDNAs that were identified using probe III we failed to isolate sequences that extended further 5' . We also found that a significant proportion of these clones (25%) ended within a 40bp region corresponding to the 5' end of the SYT sequence. When considered together with the observation that for both SYT and SYT-SSX2 the sizes of the largest cDNAs examined, respectively 3.8kb and 2.4kb, correspond to the size of the transcripts observed in Northern analyses, these results indicate that the isolated SYT and SYT-SΞX2 cDNAs represent near full length clones. The sequencing of SYT and SSX2 allows the development of PCR-based techniques for detecting the t(X,*18) translocation. When SYT and SSX2 primers were used to amplify sequences from reverse transcribed tumour RNA, PCR products of the predicted size were detected in 4/6 synovial sarcomas but not in analyses of other tumour types (Fig. 3Jb) . This observation confirms the presence of SYT-SSX2 hybrid transcripts in synovial sarcomas and provides a new method of detection of this translocation which may be of use in tumour diagnosis. In a recent examination of the t(X;18) translocation 12 the applicants have demonstrated that there is heterogeneity in the position of the X chromosome breakpoint with translocations occurring not only within the OATL2 region but also within a second OAT pseudogene region called OATL1 that maps at least 2 megabases telomeric to 0ATL2 at chromosome Xpll.2 15 . The SYT-SSX2 transcripts characterized in the present study were isolated from a synovial sarcoma containing a breakpoint at the 0ATL2 region. However the applicants found that the SSX gene probe isolated from this region also detected SSX2- related sequences within a YAC spanning the OATLl locus (Fig. 2a, lane LI.2) suggesting that t(X;18) translo¬ cations occurring at the OATLl locus also involve SSX sequences. We have also noted that rearrangements of the SYT gene were detected both in the SS255 cell line which contains a breakpoint within the OATL2 region and in the HS-SY-II cell line which has the breakpoint in the OATLl region (Fig. 2c) . These observations demonstrate that the same SYT region is involved in t(X,*18) rearrangements occurring at both the OATLl and the 0ATL2 region. The region located in OATLl is termed SSXl . The sequence for this region can be seen in fig.5. The homology between SSX2 and SSXl is sufficient that the primers designed for the SSX2 also hybridized to the SSXl region. METHODOLOGY Cell lines, tumours and somatic cell hybrids The origin and karyotype of the synovial sarcoma cell line SS255 has been described previously 9 . The A2243 synovial sarcoma line was provided by S. A. Aaronson. Both these cell lines have a t(X,*18) translocation with the breakpoint in the 0ATL2 region. The HS-SY-II cell line, characterized as described 25 , has a t(X;18) translocation with the breakpoint in the OATLl region. The human-mouse somatic cell lines Rag/255/6 and Rag/255/15 which were found by fusion synovial sarcoma 255 to mouse Rag cells carry the Xqter-pll.2;18qll.2-qter chromosome and not the reciprocal derivative or other material from chromosomes X and 18 9 . Each of these hybrid cell lines does, however, contain a selection of other human chromosomes. Tumour and blood samples from cancer patients were collected from the Royal Marsden Hospital, London and the Royal Orthopaedic Hospital, Birmingham. C12D is a human-hamster hybrid cell line containing a single human X chromosome 27 . DNA from two human-hamster cell lines containing a single copy a human chromosome 18, DL18TS and M8-S(18) , were provided by Dr N Spurr and Prof R Newbold. YAC clones YAC clones were isolated and characterized as described previously 11 . Clones OATL2.7 and OATL2.10 corresponding to the OATL2 locus contain inserts of 450kb and 300kb 27 - respectively. Clone OATLl.2 corresponding to the OATLl locus contain an insert size of 650kb. Total DNA from yeast strains harbouring individual YAC clones was prepared as described 11 . To isolate purified YAC DNA agarose blocks of DNA were prepared from yeast spheroblasts as described 11 and loaded onto 1% (w/v) low melting point agarose gels containing 0.25 x TBE gel (5 x TBE contains 0.9m Tris-borate, 2mM EDTA) . Electropho- resis was performed at 210V in Rotaphor apparatus (Biometra) for 20h at 13 °C using log-ramped pulse times (80-20 seconds) and electrode angle (115°-90°) . The DNA was stained with ethidium bromide and visualised on a UV transilluminator. YAC DNA which appeared as a single extra band was excised and radiolabelled directly. Analysis of DNA and RNA Preparation and analysis of genomic DNA and cytoplasmic RNA by, respectively, Southern and Northern analysis was carried out exactly as described previously 6,28,29 . Equal loading of samples was checked by staining with ethidium bromide and visualization of DNA and RNA under UV light. Radiolabelling of DNA probes was performed using [a- 32 P] dCTP (3000 Ci mM "1 , Amersham) by the method of Feinberg and Vogelstein 30 . Hybridization of Southern and Northern blots to radiolabelled probes and washing of blots was also carried out exactly as described previously 6,28,29 . Autoradiography was conducted at -70 °C for 1-3 days using Fuji RX film and an intensifying screen. Size markers for Southern blots were BRL kilobase ladder and BRL high molecular weight DNA markers. The BRL 0.24-9.5kb ladder was used as RNA size markers. An SSXl polynucleotide sequence may be distinguished from an SSX2 polynucleotide sequence by use of the restriction enzymes Lsp and Smal. Lspl cuts only SSXl polynucleotide originating sequences and not SSX2 polynucleotide originating sequences. Likewise Smal cuts only SSX2 28 SUBSTITUTE SHEET (RULE 2 polynucleotide originating sequences and not SSXl polynucleotide originating sequences. RT-PCR analyses lμg of total RNA was reverse transcribed using Superscript II reverse transcriptase (GIBCO BRL) and random primers according to recommended conditions. The resulting cDNA was subject to amplification using two sets of primers. To test for the presence of the SYT-SSX hybrid mRNA, amplification was carried out with primers corresponding to positions 949-968 (forward primer) in SYT and 1512-1531 (reverse primer) in SSX2 (Fig. 4) . As a positive control to confirm that each RNA sample could yield products after RT-PCR, amplification was carried out with two actin primers 5' -GAGCGGGAATCGTGCGTGACATT- 3 'and 5' -GATGGAGTTGAAGGTAGTTTCGTG-3 ' from separate exons that should amplify a 234bp cDNA actin sequence. In these analyses all reverse transcribed samples gave an actin PCR product of the expected size. The amplification conditions were 93°C for 1 min, 55°C for lmin and 72°C for 1 min for .30 cycles in a final volume of 25μl. The products were separated by electrophoresis in agarose gels followed by staining with ethidium bromide. cDNA and genomic DNA libraries cDNA libraries were made from poly A* RNA from A2243 cells in the XCEV29 vector as described previously 31,32 . Screening of this cDNA library was carried out as described by Snell et al . 33 following plating the library at low density (16,000 plaques per 22 x 22 cm plate) and allowing growth to continue until the plaques were almost confluent. The efficiency of detection of cDNA clones using YACs as probes was high because using the 450kb YAC OATL2.7 which contains OAT pseudogene sequences it was possible to detect 80% of the clones that were detected using an OAT cDNA probe. A cDNA library prepared from M426 human fibroblast cells was obtained from S.A. Aaronson. This library was plated at a density of 30,000 plaques/plate. Plaque lifts were carried out using Hybond N (Amersham) and Zeta probe (Bio-rad) according to the manufacturers protocol. Genomic DNA libraries were made by inserting DNA from the Rag/225/15 somatic cell hybrid cell line that had been partially digested with Sau3A into the BamHl site of the EMBL4 vector as described previously 31 . Genomic DNA libraries were also screened as described previously 31 . DNA sequencing cDNA inserts were sequenced by the dideoxy method 34 using the Sequenase version 2 sequencing kit (Amersham) after subcloning and exonuclease III deletion of selected restriction fragments in the phagemids KS * , KS " , SK * and SK " (Strategene) and rescuing single-stranded templates a described by the supplier. Sequence of both strands of SYT and SYT-SSX clones was completed using this method. Figs. 4b and 5 are respectively in respect of the human SYT-SSX2 and SYT-SSXl cDNA nucleotide and predicted amino acid sequences . In these fusion constructs part of the SSX sequences have been substituted with SYT sequences. However the provision by the present applicants of a substantial portion of the SSXl and SSX2 sequences allows the skilled person to identify, obtain and sequence entire SSXl and SSX2 sequences by the use of standard methodologies and procedures eg by designing probes based on the sequence information provided herein to screen a suitable library eg a testis tissue library (see also below) . Normal SSXl and SSX2 Figure 7 gives the SSXl and SSX2 cDNA nucleotide and predicted amino acid sequences. RNA was extracted from a selection of human tissues and tumour cell lines and examined by Northern analyses using an SSX probe under hybridization conditions that would detect both SSXl and SSX2 transcripts. These studies identified discrete transcripts of 1.6 kb in normal testis and in the HT1080 fibrosarcoma cell line. In addition a more diffuse band in the size range 1.4-1.6 kb was detected in A673 rhabdomyosarcoma cells and at low levels in normal thyroid. To characterise the transcripts of the normal SSXl and SSX2 genes a 3 ' SSX2 gene probe was used to screen cDNA libraries prepared from RNA extracted from the HT1080 cell line and from human testis. Sequencing of the clones isolated from the HT1080 cDNA library produced a continuous sequence of 766 bp that contained an open reading frame of 188 amino acids. The 3' region of this sequence exactly matched the SSXl sequences identified in the SYT-SSXl fusion transcript from the HS-SY-II cell line indicating that the HT1080 cDNA clones correspond to transcripts of the normal SSXl gene. The cDNA clones isolated from the human testis cDNA library correspond exactly to the SSX2 sequence present in the SYT-SSX2 fusion transcript from the A2243 cell line. Since these clones were not full length, 5' RACE and PCR amplification were used to isolate the remaining 5' SSX2 sequences from reverse-transcribed testis RNA. The normal SSX2 sequence obtained in these studies has an open reading frame encoding a protein of 188 amino acids which exhibits a high extent of homology (81% identify) to the 188 amino acid, SSXl protein (Figure 7) . Other common features of the SSXl and SSX2 proteins include the abundance of charged amino acids (40-41%) and the presence of consensus sequences for N-glycosylation and tyrosine phosphorylation. It is also notable that both the SSXl and SSX2 proteins contain acidic C-terminal tails in common with several nuclear proteins such as the HMG proteins, and yeast S1N1. Different types of SYT-SSX transcripts To investigate the degree of variability in the t(X;18) breakpoint, RNA from a series of 32 synovial sarcomas and cell lines was reverse-transcribed and subjected to PCR with SYT and SSX primers . PCR products were detected in 29 of the tumours. In the three tumours which did not yield PCR products the applicants failed to detect either abnormal SSX transcripts by Northern analysis or rearrangement of the SYT gene by Southern analysis. Sequencing of the PCR products revealed that in 26 tumours, the junction between SYT and SSX occurred at the same position as the SYT-SSXl and SYT-SSX2 breakpoints described above for the HS-SY-II and A2243 cell lines respectively. The uniform structure of these transcripts agrees with the breakpoint being present in specific introns of the SYT, SSXl and SSX2 genes. Three tumours containing variant transcripts were detected. In tumour STS498 the SYT-SSXl transcript contained an additional 144 bp of normal SSXl sequence but had not lost 132 bp of SYT sequence, indicating that heterogeneity in the position of the breakpoint can occur within both the SYT and SSXl genes. Tumours STS 159 and STS255 contained a second variant transcript in which there was an insert of 87 bp between previously identified SYT and SSX sequences (see Fig. 6) . Mapping by PCR and Southern analyses using OTAL1 and OATL2 YACs and somatic cell hybrid lines containing individual human chromosomes demonstrated that this sequence is derived from the X chromosome (data not shown) . The 87 bp sequence did not, however, match the sequences present in the normal SSX2 transcripts and could not be detected by RT-PCR in cell lines and tissues that expressed normal SSXl and SSX2 transcripts suggesting that it may represent a cryptic exon that is derived from SSX2 intronic sequences. The generation of a cryptic exon from intronic sequences has also been documented for the EWS-FLII gene rearrangement observed in Ewings sarcoma. REFERENCES 1. Enzinger, F.M. & Weiss, S.W. Soft Tissue Tumours, 2nd edn. Mosby, St Louis (1988) . 2. Smith, S., Reeves, B.R., Wong, L. & Fisher, C. 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Stroeher, V.L., Jorgensen, E.M. & Garber, R.L. Multiple transcripts from the Antennapedia Gene of Drosophilia melanogaster. Mol . Cell Biol . 6, 4667- 4675 (1986) . 22. Mylin, L.M. , Gerardot, C.J., Hopper, J.H. _. Dickson, R.C. Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests, functional domains. Nuc . Acids Res . 19, 5345-5350 (1991) . 23. Buettner, R., Yin, S.O., Hong, Y.S., Boncinelli, E. & Tainsky, M.A. Alteration of homeobox gene expression by Ν-ras transformation of PA-1 human teratocarcinoma cells. Mol . Cell . Biol . 11, 3573- 3583 (1991) . 24. Tamkun, J.W. et al. brahma : a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SW12. Cell 68, 561-571 (1992) . 25. Sonobe, H., et al. Establishment and characterization of a new human synovial sarcoma cell line HS-SY-II. Lab. Invest. 67, 498-505 (1992) . 26. Fisher, C. Synovial sarcoma. Current Diagnostic Pathology, 1, 13-18 (1994) . 27. Gross, S.J. & Harris, H. New method for mapping genes in human chromosomes. Na ture 255, 68-683 (1975) . 28. Mitchell, P.J. _. Cooper, C.S. The human tpr gene encodes a protein of 2094 amino acids that has extensive coiled-coil regions and an acidic C- terminal domain. Oncogene 7, 2329-2333 (1992) . 29. Mitchell, P.J. & Cooper, C.S. Nucleotide sequence analysis of human tpr cDNA clones. Oncogene 7, 383- 358 (1992) . 30. Feinberg, A.P. & Vogelstein, B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal . Biochem. 132, 6-13 (1983) . 31. Cooper, C.S. et al. Molecular cloning of a new transforming gene from a chemically-transformed human cell line. Na ture 311, 29-33 (1984) . 32. Mikie, T. et al . Development of a highly efficient expression cDΝA cloning system: application to oncogene isolation. Proc . natn. Acad. Sci. U.S.A. 88, 5167-5171 (1991) . 33. Snell, R.G. et al. 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Claims: CLAIMS 1. A method for diagnosing the presence or absence of synovial sarcoma which comprises obtaining a sample derived from a tissue of a subject,* contacting the sample with a specific binding member (sbm) which is specific for a binding partner (bp) which is characteristic of synovial sarcoma such that the presence of bp in the sample is diagnostic of synovial sarcoma, the bp being absent from an equivalent sample derived from a subject without synovial sarcoma,* and detecting any binding of the sbm to its bp. 2. A method according to claim 1 wherein the sbm comprises nucleic acid. 3. A method according to claim 1 or claim 2 wherein the bp is an oligo- or polynucleotide sequence characteristic of an SYT, SSXl or SSX2 polynucleotide sequence, or of an SYT-SSXl or an SYT-SSX2 polynucleotide sequence resulting from a translocation event. 4. A method according to any one of claims 1 to 3 wherein the sbm comprises a pair of oligonucleotide primers for amplification of a sequence characteristic of an SYT, SSXl, SSX2, SYT-SSXl or SYT-SSX2 polynucleotide sequence. 5. A method according to claim 4 wherein the bp is an SYT-SSXl or an SYT-SSX2 polynucleotide sequence resulting from a translocation event and one primer binds to a portion of the SYT sequence and the other primer binds to a portion of the SSX sequence. 6. A method according to any one of claims 1 to 5 wherein the sbm comprises an oligo- or polynucleotide sequence hybridizable to (a) a polynucleotide sequence which codes for part or all of an amino acid sequence as shown in either of Fig. 4(a) or Fig. 7; (b) a polynucleotide sequence which codes for part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4(b) ; or (c) a polynucleotide sequence which codes for part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 5. 7. A method according to claim 6 wherein the oligo- or polynucleotide sequence comprises part or all of the polynucleotide sequence shown in either Fig. 4(a) , Fig. 4(b) , Fig. 5, or Fig. 7 or a sequence complementary thereto. 8. A method according to claim 1 wherein the sbm comprises an antibody binding domain. 9. A method according to claim 8 wherein the bp is an oligo- or polypeptide sequence characteristic of an SYT, SSXl or SSX2 polypeptide sequence or of an SYT-SSXl or an SYT-SSX2 polypeptide sequence resulting from a translocation event. 10. A method according to claim 8 or claim 9 wherein the sbm comprises an antibody binding domain for (a) an oligo- or polypeptide sequence which comprises part or all of an amino acid sequence as shown in either of Fig 4(a) or Fig. 7; (b) an oligo- or polypeptide sequence which comprises part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4 (b) ,* or (c) an oligo- or polypeptide sequence which comprises part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig 5. 40 SUBSTITUTE SHEπ- /n ni c 11. A method according to claim 1 wherein the bp is an antibody. 12. A method according to claim 11 wherein the sbm comprises an oligo- or polypeptide sequence characteristic of an SYT, SSXl or SSX2 polypeptide sequence or of an SYT-SSXl or an SYT-SSX2 polypeptide sequence resulting from a translocation event. 13. A method according to claim 11 or claim 12 wherein the sbm comprises (a) an oligo- or polypeptide sequence which comprises part or all of an amino acid sequence as shown in either of Fig 4(a) or Fig. 7; (b) an oligo- or polypeptide sequence which comprises part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig 4 (b) ; or (c) an oligo- or polypeptide sequence which comprises part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 5. 14. A method according to any one of claim 1 to 13 wherein the bp is absent from tumours other than synovial sarcoma. 15. An sbm for use in a method according to claim 1 which comprises an oligo or polynucleotide sequence for a bp which is an oligo- or polynucleotide sequence characteristic of an SAT, SSXl or SSX2 polynucleotide sequence or an SAT-SSX1 or SAT-SSX2 polynucleotide sequence resulting from a translocation event. 16. An sbm according to claim 15 which comprises a pair of oligonucleotide primers substantially complementary to a pair of target sequences within an SYT, SSXl or SSX2 polynucleotide sequence, which target sequences are spaced apart from one another. 17. An sbm according to claim 15 which comprises a pair of oligonucleotide primers substantially complementary to a pair of target sequences within a SYT-SSXl or SYT-SSX2 polynucleotide sequence and one primer binds to a target in the SAT sequence and the other primer to a target in the SSX sequence, which target sequences are spaced apart from one another. 18. An sbm for use according to any one of claims 15 to 17 which comprises an oligo or polynucleotide sequence hybridizable to (a) a polynucleotide sequence which codes for part or all of an amino acid sequence as shown in either of Fig. 4(a) or Fig. 7; (b) a polynucleotide sequence which codes for part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4(b) ; or (c) a polypeptide sequence which codes for part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 5. 19. An sbm according to claim 18 wherein the oligo- or polynucleotide sequence comprises part or all of the polynucleotide sequence shown in either Fig. 4(a), Fig. 4(b) Fig. 5, or Fig. 7 or a sequence complementary thereto. 20. An sbm for use in a method according to claim 1 which comprises an antibody binding domain. 21. An sbm according to claim 20 wherein the antibody binding domain is for an oligo- or polypeptide sequence characteristic of an SYT, SSXl, SSX2, SYT-SSXl or SYT- SSX2 polypeptide sequence. 22. An sbm according to claim 20 or claim 21 wherein the antibody binding domain is for either (a) an oligo- or polypeptide sequence which comprises part or all of an amino acid sequence as shown in either of Fig. 4(a) or Fig. 7; (b) an oligo- or polypeptide sequence which comprises part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4(b) ; or (c) an oligo- or polypeptide which has part or all of the amino acid sequence shown in Fig. 5. 23. An sbm for use in a method according to claim 1 which comprises an oligo- or polypeptide sequence characteristic of an SYT, SSXl, SSX2, SYT-SSXl or SYT- SSX2 polypeptide sequence. 24. An sbm according to claim 23 which comprises (a) an oligo- or polypeptide sequence which comprises part or all of an amino acid sequence as shown in either of Fig. 4 (a) or Fig. 7; (b) an oligo- or polypeptide sequence which comprises part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4 (b) ; or (c) an oligo- or polypeptide sequence which comprises part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 5. 25. A nucleic acid which comprises an oligo- or polynucleotide sequence characteristic of an SYT, SSXl or SSX2 polynucleotide sequence or of an SYT-SSXl or SYT- SSX2 polynucleotide sequence resulting from a translocation event. 26. A nucleic acid according to claim 25 which comprises an oligo- or polynucleotide sequence hybridizable to (a) a polynucleotide sequence which codes for part or all of an amino acid sequence as shown in either of Fig. 4(a) or Fig. 7; (b) a polynucleotide sequence which codes for part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4(b) ; or (c) a polynucleotide sequence which codes for part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 5. 43 SUBSTITUTE SHΓ r-r ,nt .ULE 27. A nucleic acid according to claim 26 wherein the oligo- or polynucleotide sequence comprises part or all of the polynucleotide sequence shown in either Fig. 4(a) , Fig. 4(b) , Fig. 5 or Fig. 7 or a sequence complementary thereto. 28. A vector comprising nucleic acid according to any one of claims 25 to 27. 29. A vector according to claim 28 which is a recombinant transfer vector. 30. A vector according to claim 28 which is an expression vector. 31. A vector according to claim 28 which is a vaccine vector. 32. A vector according to claim 31 which is from a pox virus. 33. A vector according to claim 32 which is from a vaccinia virus. 34. A recombinant host cell which comprises a vector according to any one of claims 28 to 33. 35. An oligo- or polypeptide sequence characteristic of an SYT, SSXl or SSX2 polypeptide sequence or of an SYT- SSXl or SYT-SSX2 polypeptide sequence resulting from a translocation event. 36. An oligo- or polypeptide sequence according to claim 35 which comprises (a) part or all of an amino acid sequence as shown in either of Fig. 4(a) or Fig. 7; (b) part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4(b) ; or (c) part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 5. 37. An oligo- or polypeptide according to claim 36 which is immunologically cross-reactive with a polypeptide having the amino acid sequence of either Fig. 4(a), Fig. 4(b), Fig. 5, or Fig. 7. 38. A polypeptide sequence according to claim 35 or claim 36 which comprises part or all of the amino acid sequence of either Fig. 4a, Fig. 4(b), Fig. 5 or Fig. 7. 39. A molecule comprising an antibody binding domain for an oligo- or polypeptide sequence characteristic of an SYT, SSXl SSX2, SYT-SSXl or SYT-SSX2 polypeptide sequence. 40. A molecule according to claim 39 wherein the antibody binding domain is for either (a) an oligo- or polypeptide sequence which comprises part or all of an amino acid sequence as shown in either of Fig. 4(a) or Fig. 7; (b) an oligo- or polypeptide sequence which comprises part or all of a polypeptide which has part or all of the amino acid sequence shown in Fig. 4(b); or (c) an oligo- or polypeptide which has part or all of the amino acid sequence shown in Fig. 5. 41. A molecule according to claim 39 or claim 40 which is an antibody. 42. A molecule according to claim 41 which is a monoclonal antibody. 43. A molecule according to claim 42 which is a humanised monoclonal antibody. 44. A pharmaceutical which comprises as an active ingredient a molecule according to any one of claims 39 to 43 or a vector according to any one of claims 31 to 33. 45. Use of a molecule according to any one of claims 39 to 43 or a vector according to any one of claims 31 to 33 in the preparation of a medicament for the prophylaxis or treatment of synovial sarcoma. 46. A method of preventing or treating synovial sarcoma which comprises administering to a patient a pharmaceutical according to claim 44. 47. An oligo- or polynucleotide which can be transcribed to produce an antisense oligo- or polynucleotide which is at least in part complementary to an SYT, SSXl, SSX2, SYT-SSXl or SYT-SSX2 mRNA sequence and wherein the complementary portion of the antisense oligo- or polynucleotide is of sufficient length to inhibit translation of a said mRNA and production of a polypeptide encoded by the mRNA. 48. A pharmaceutical which comprises as an active ingredient an oligo- or polynucleotide according to claim 47. 49. Use of a oligo- or polynucleotide according to claim 47 in the preparation of a medicament for the treatment of synovial sarcoma. 50. A method of treating a patient with synovial sarcoma which comprises administering to the patient a pharmaceutical according to claim 48. 51. A method according to claim 4 or claim 5 which also comprises: contacting the sample with ingredients for carrying out a polynucleotide sequence amplification; applying standard hybridization, elongation and denaturation conditions; and detecting the binding of sbm to its bp by looking for the presence or absence of an amplification product.

Inventors: COOPER COLIN STEPHEN, GUSTERSON BARRY AUSTIN, COOPER, COLIN, STEPHEN, GUSTERSON, BARRY, AUSTIN
Applicants: CANCER RES CAMPAIGN TECH, COOPER COLIN STEPHEN, GUSTERSON BARRY AUSTIN, CANCER RESEARCH CAMPAIGN TECHNOLOGY LIMITED, COOPER, COLIN, STEPHEN, GUSTERSON, BARRY, AUSTIN
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, C07K14, C12N15, C12Q1