Detection of fusion genes and fusion proteins in sarcoma : methodological and clinical aspects

Sammanfattning: Ewing's sarcoma/PNET and synovial sarcoma, all regarded as high-grade tumours, have their peak incidence during the second decade of life, thus affecting children and adolescents. Both tumour types have specific chromosomal translocations, i.e. t(11;22) and t(X;18), respectively, resulting in fusion genes coding for chimeric proteins. The t(11;22) translocation, present in 85% of Ewing's sarcoma/PNET, results in the fusion of the EWS gene to the FLI- 1 gene forming EWS-FLI- 1 and the synovial sarcoma t(X;18) translocation fuses the SYT gene to either SSX1 or SSX2 resulting in SYTSSX1 or SYT-SSX2. These translocations are believed to play a causative role in the oncogenic differentiation. As a consequence of the alternative breakpoints on the involved chromosomes different variants of the fusion genes exist. This thesis aims to optimise molecular biology methodologies for the detection of fusion genes and fusion proteins in Ewing's sarcoma/PNET and synovial sarcoma from fine needle aspirates, fresh frozen biopsies and formalin-fixed paraffinembedded archive material and to evaluate the relevance of the different variants of synovial sarcoma fusion gene transcripts regarding morphology, proliferation, and prognosis in synovial sarcoma. A polyclonal antibody raised against the C-terminal of murine FLI-1 was used to detect the EWSFLI-1 fusion protein by Western blotting and immunohistochemistry. When applied to Ewing's sarcoma tumour samples the EWS-FLI-1 protein was detected by Western blotting in cases with the t(11;22) translocation. Immunohistochemically 80 percent (12 out of 15 cases) of the Ewing's tumours exhibited a positive immunoreactivity for the FLI-1 antibody. Two types of small round cell tumours (neuroblastoma and alveolar rhabdomyosarcoma) were negative. Since the t(11;22) translocation is present in approximately 85% of the Ewing' sarcoma/PNET cases Western blotting and immunohistochemistry can be used as an adjunctive diagnostic tools. Reverse transcription-polymerase chain reaction (RT-PCR) was applied to fine needle aspirates without prior RNA extraction. This proved to be a sensitive method since the lowest amount of tumour material needed to obtain a detectable result was ten tumour cells. The expressed SYT-SSX chimeric mRNA transcripts were rapidly detected by RT-PCR. In several cases with uncharacteristic morphological features, the SYT-SSX gene fusion was verified using RT-PCR. The potential clinical relevance of SYT-SSX1 and SYT-SSX2 fusion transcripts together with tumour proliferation was investigated. In a series of 33 patients with primary synovial sarcoma the type of fusion transcript was assessed by reverse transcription-polymerase chain reaction and sequence analysis. Proliferation rate was analysed using anti-Ki-67 antibodies. The hazard ratio (HR) with respect to metastasis-free survival for patients with SYT-SSX1 vs. patients with SYT-SSX2 fusion transcripts was 7.4 (95% CI 1.5-36, log-rank P=0.004), meaning that patients with SYT-SSX1 transcripts had an impaired clinical outcome. The 5-year metastasis-free survival for patients with SYT-SSX1 was 42% vs. 89% for patients with SYT-SSX2. There was a significant association between SYT-SSX1 and high tumour proliferation rate (P=0.02). In conclusion, the findings suggest that the type of SYT-SSX1 fusion transcript determines the proliferation rate and may be an important predictor of clinical outcome for patients with synovial sarcoma. In addition, a novel fusion gene, SYT-SSX4, was identified in a case of typical synovial sarcoma. This study shows that adjunctive techniques can detect fusion genes and fusion proteins and that variant gene fusion defines clinically different phenotypes.