1. J. Fernebro, P. Francis, P. Edén, Å. Borg, I. Panagopoulos, F. Mertens, J. Vallon-Christersson, M. Åkerman, N. Mandahl, M. Nilbert Departments of Oncology, Theoretical Physics, Clinical Genetics, and Pathology Lund University, Sweden Princy.Francis@onk.lu.se Gene expression profiles relate to gene fusion type and metastasis in synovial sarcoma

2. Aims Groups of genes discriminating the monophasic from biphasic have been previously identified (Allander et al., 2002; Nagayama et al., 2002) Do gene expression profiles in synovial sarcoma correlate with:  Histopathology  Cytogenetics  Gene fusion type  Metastatic potential

3. 23 synovial sarcoma specimens from 21 patients mean age 46, 12 males, 16 primary tumors, 3 local recurrences, 4 metastases  Histopathology monophasic (17) vs biphasic (4)  Cytogenetics t(X;18) (4) vs complex karyotype (12)  Gene fusion type SS18/SSX1 (12) vs SS18/SSX2 (7)  Metastatic potential metastasis (13) vs metastsis-free (5) SS18 SSX Material

4. Scan Process Image Labelled cDNA 18-20 hrs incubation at 42 o C Hybridize to microarray slide Reference RNA Tumour RNA Image and Data Analysis cDNA microarray, 27648 IMAGE clones 25648 distinct reporters 17517 unique UniGene clusters Hierarchical clustering based on ~5800 genes - Unsupervised - Supervised Golub scores, random permutation tests Methods

5.  Expression profiles did not relate to histopathology or karyotype  Significant discriminators for gene fusion type and metastatic potential Metastasis  No  Yes Gene fusion type  Type 1  Type 2 Supervised clustering

6. Up SS18/SSX1 - TCF7- LEF1 - RTP801- CKB - NOTCH2- Metallothioneins Up SS18/SSX2 - BENE- MN1 - NEDD4- FRAG1 - MSX2- TIEG Gene fusion type  Transcription factors  Signalling molecules  Multiple metallothioneins Discriminating genes Up metastasis - TYMS- TOP2A - PTTG1- IGFBP2 - BUB1- COL18A1 Up without metastasis - ProSAPiP1- TNNT1 - ICAM1- DDX21 - FRAG1- TCF8 Metastatic potential  Cell proliferation  Cell cycle progression  Transcriptional regulation

7.  45 from top 200 common to both lists  Up in metastasis also up in SS18/SSX1 - BSG- FRAG1 - NOTCH2- RGS2 - TCF7- TCF8 Common discriminators Up SS18/SSX1 - TCF7- LEF1 - RTP801- CKB - NOTCH2- Metallothioneins Up SS18/SSX2 - BENE- MN1 - NEDD4- FRAG1 - MSX2- TIEG Gene fusion type Up metastasis - TYMS- TOP2A - PTTG1- IGFBP2 - BUB1- COL18A1 Up without metastasis - ProSAPiP1- TNNT1 - ICAM1- DDX21 - FRAG1- TCF8 Metastatic potential Discriminating genes

8. Targets for chemotherapeutic drugs Up SS18/SSX1 - TCF7- LEF1 - RTP801- CKB - NOTCH2- Metallothioneins Up SS18/SSX2 - BENE- MN1 - NEDD4- FRAG1 - MSX2- TIEG Gene fusion type Up metastasis - TYMS- TOP2A - PTTG1- IGFBP2 - BUB1- COL18A1 Up without metastasis - ProSAPiP1- TNNT1 - ICAM1- DDX21 - FRAG1- TCF8 Metastatic potential Discriminating genes

9. Previously reported in synovial sarcoma Up SS18/SSX1 - TCF7- LEF1 - RTP801- CKB - NOTCH2- Metallothioneins Up SS18/SSX2 - BENE- MN1 - NEDD4- FRAG1 - MSX2- TIEG Gene fusion type Up metastasis - TYMS- TOP2A - PTTG1- IGFBP2 - BUB1- COL18A1 Up without metastasis - ProSAPiP1- TNNT1 - ICAM1- DDX21 - FRAG1- TCF8 Metastatic potential Discriminating genes

10. Previously reported in metastasizing tumors Up SS18/SSX1 - TCF7- LEF1 - RTP801- CKB - NOTCH2- Metallothioneins Up SS18/SSX2 - BENE- MN1 - NEDD4- FRAG1 - MSX2- TIEG Gene fusion type Up metastasis - TYMS- TOP2A - PTTG1- IGFBP2 - BUB1- COL18A1 Up without metastasis - ProSAPiP1- TNNT1 - ICAM1- DDX21 - FRAG1- TCF8 Metastatic potential Discriminating genes

11.  Differentially expressed genes relative to gene fusion type and metastatic potential in synovial sarcoma  Signaling molecules, transcription factors and metallothioneins among the fusion type discriminating genes  Known targets for chemotherapy (TYMS and TOP2A) upregulated in metastasizing tumors Conclusions