1 5 2 A b s t r a c t s

IMMUNODETECTION OF THE SYNOVIAL SARCOMA-ASSOCIATED SYT AND SSX PROTEINS

CYTOGENETIC CHARACTERIZATION OF A CLIVUS CHORDOMA IN A CANCER FAMILY PATIENT

NR. dos Santos, D. de Bruijn, B. de Leeuw, A. Geurts van Kessel

Department of Human Genetics, University Hospital Nijmegen, The Netherlands

R. Malgara, L. DoIDr6, P. Riva, M. Volont~, L. Larizza, AM. Fuhrmon Conti

Dipartimento di Biologio e Genetica per le Scienze Mediche, UniversitO di Milano, Italia

Synovial sarcomas are characterized by the occurence of a specific chromosomal translocation, t(X; 18)(p 1 i .2;q I 1.2). Positional cloning studies have revealed that in these tumors the SYT gene on chromosome 18 fuses to either one of the SSXI or the SSX2 genes, both of which are located on the X chromosome. The SYT gene encodes a protein rich in glutamines, prolines and glycines with no significant homology to any known sequence, notwithstanding the presence of putative SH3- and SH2-binding domains. The proteins encoded by the SSX1 and SSX2 genes are highly homologous and both contain Kriippel-associated boxes (KRABs), previously identified in a group of zinc finger proteins. However, up till now the exact functions of the SYT and SSX proteins have remained unclear. In order to obtain further clues about these functions, we generated antibodies against the SYT and SSX proteins. Therefore, a sequence of 492 bp from SSX2 was cloned into a pGEX prokaryotic expression vector and a fusion protein (GST-SSX2) was purified after expression in E. coli. Subsequently, this protein was injected subcutaneously into rabbits and a polyclonal antiserum against SSX2, as assessed by immunoblotting, was obtained. Similarly, a rabbit polyclonal antiserum against an in vitro synthesized SYT (264-277 aa) peptide was developed. These specific antibodies directed against the synovial sarcoma- associated SYT and SSX proteins are currently used to perform immunohistochemical and immunofluorescent studies on normal or tumor tissues. The results from these studies will be presented in detail.

ADDITIONAL HUMAN SSX GENES NOT INVOLVED IN THE t (X;18) (pl 1.2; ql 1.2) AND MOLECULAR DIAGNOSIS OF SYNOVIAL SARCOMAS

B. de Leuuw ~, M. Balemans I , A. Geurts van KesseP

IDeportment of Human Genetics, University Hospital Nijmegen, The Netherlands

Cytogenetic analysis of chordoma, a rare malignancy which develops at multiple sites is so far restricted to 14 cases (1-2). We report the cytogenetic findings on a cranial chordoma in a 39 year old male patient who manifested it first at age 8 and underwent radiotherapy thereafter. Interestingly his second daughter developed an astrocytoma at age 11 and his third daugther presented with a cranial chordoma at age 5. A normal karyotype was observed in all family members and no fragile sites were found to be expressed. Cytogenetic analysis performed on the tumor specimens from the first surgery after collagenase treatment and 72 hours of culture evidenced a modal chromosome number of 40-41 in a total of 42 QFQ-banded metaphases. A t(1;9)(p36.1; p21) was found in 80% of the metaphases, while two other clonal rearrangements, add6q and addl(pl l) were observed in 10% of the cells. HSRs involving 7q, 10p, l 2p, 12q, 17p were observed sporadically.

One year from the surgical removal the tumor regrew at the same site and a biopsy was obtained before surgery. Cytogenetic analysis on the bioptic material showed the recurrence of the t(l;9) and the add6q previously recorded.

The patient underwent a new surgery and tumor specimens were collected for cytogenetic and molecular analysis aimed at establishing the causal role of t(1 ;9) in chordoma development.

References 1- Mertens F et al Cancer Genet Cytogenet 1994; 73:147-151. 2- Bridge JA et al Cancer Genet Cytogenet 1994; 75:23-25

During the past decade, the status of the (X; 18) (p 11.2; q 11.2) translocation as the eytogenetic hallmark for synovial sarcoma has clearly been established (l).

The isolation by positional cloning of the t (X;18) (pl 1.2; q l l . 2 ) chromosomal breakpoint, characteristic for synovial sarcoma, has been described by us, followed by the description of the genes involved, SYT and SSX (2,3). In the course of these studies we found that two alternative X- chromosomal breakpoint regions can be involved in different tumor subtypes.

The genes which are affected by these two translocations, SYT, SSX1 and SSX2, and the corresponding fusion products, SYT-SSX1 and SYT-SSX2 respectively, have been isolated and characterized. Here we report the existance of additional genes homologous to SSXI and SSX2, which do not seem to be involved in ( X; 18) translocations.

Furthermore, we present an example of how these results can be used to assist synovial sarcoma diagnosis, using RT-PCR and Northern blotting.

References 1) Turc-Care|, C. etal., Proc. Natl. Acad. Sci. USA 84 : 1981-1985 (1987) 2) Clark, J. etal., Nature Genet. 7:502-508 (1994) 3) Crew, A.J. et al., EMBO J. 14:2333-2340 (1995)