Primary Synovial Sarcoma (SS) of the digestive system: a molecular and clinicopathological study of fifteen cases

Fifteen cases of SS of the abdominal viscera were collected from three Italian institutions, the consultation files of one of the Authors (APDT) and Departments of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA. Patients’ clinical records were retrieved. Follow-up information was available for 11 patients (Table 1). All samples were handled in a coded fashion, and all procedures were performed according to the ethical guidelines of the local institutions.

All the cases were reviewed for diagnostic confirmation and both necrosis extent and mitoses count evaluated, grading provided according to French National Federation of Cancer Centers (FNLCC) (Table 2). In a subset of cases neoadjuvant chemotherapy was applied and pre-chemo biopsies were not available (Table 2); the values for mitoses and necrosis are no longer relevant for these cases as pre-treated specimens cannot be accurately graded under the FNCLCC system.

Immunostaining was performed for EMA, cytokeratin AE1/AE3, SMA, Desmin, CD34, CD117, S-100, CD99, CD56 and TLE1. Four-μm sections of formalin-fixed paraffin-embedded material were used according to standard laboratory procedures. Details of the antibodies used are given in Table 3.

For case 14 conventional karyotype was performed. Cell culture, harvest conditions, and karyotyping were performed according to standard protocols.

Hybridisation was performed according to the manufacturer’s protocol. Slides were mounted and counterstained with anti-fade DAPI (Vysis, Downers Grove, IL, USA), visualized using an epifluorescent microscope (Olympus BX61) and analysed with FISH analysis software (Genetix-Cytovision 4.5.1). 300 interphase nuclei were analyzed.

Ten to fifteen 15 μm-thick sections from paraffin-embedded tissue were de-paraffinized twice using xylene, washed twice with absolute ethanol followed by TNE1X, resuspended in 250 μl of ATL buffer (Qiagen) with the addition of proteinase K (Qiagen), and incubated for 72 hours at 55°C under moderate shaking. The percentage of tumor cells, as calculated from the HE-stained slides, was at least 70%. Subsequently, RNA was extracted with TRIzol-LS Reagent (Gibco BRL), according to the manufacturer’s instructions. RNA pellets were resuspended in 10–20 μl of RNAse-free water and stored at −80°C. 5 μg of total RNA were reverse transcribed in a total volume of 20 μl using specific reverse primers for SSX and BETA2M genes, respectively. Samples were incubated at 42°C for 1 hour, then at 72°C for 15 minutes. PCR amplification of each sample and a 1:20 dilution were performed in duplicate using 96-well plates in 25 μl reaction mixture containing 300 nM of each primer, 200 nM of each probe (SSX1-SSX2) or 100 nM probe BETA2M and 2X TaqMan Gene Expression Master Mix (Applied Biosystems, CA), on Applied Biosystems StepOnePlus Real-Time PCR Systems (Applied Byosystem). (For primer and probe sequences, see Table 4). Thermal cycling conditions were 2 minutes at 50°C, 10 minutes at 95°C, then 50 cycles for three PCR steps consisting of 30 seconds at 95°C and 1 minute at 60°C. Eight cases were studied by Q-PCR, for all of them a positive control product was amplified.