Ewing sarcoma is a malignant bone tumour characterized, in 90% of the cases, by the balanced chromosomal translocation t(11;22) which generates a chimeric oncogene that acts as a transcriptional activator. The detection of translocation can be fundamental in cases with an extraosseous or unusual location which are histologically difficult to diagnose and it is also helpful in evaluation of residual disease. We joined immunohistochemical analysis and routine RT-PCR method together, the latter one allowing the detection of the most common fusion transcript EWS-FLI1 in archival paraffine-embedded tissues of EFT patients. We used a pair of primers which allowed us to discriminate between two subtypes of EWS-FLI1 transcript. We selected some sample for EWS-FLI1 typing using a Real-Time PCR assay.

We analysed 54 EFT patients. RNA was extracted from paraffine-embedded sections and reverse transcribed into cDNA. On every sample we performed RT-PCR and immunohistochemistry for the marker CD99; we also selected 5 samples for Real-Time PCR analysis.

Fourty-nine out of 54 samples had a RNA suitable for analysis. Thirty-six patients had EWS-FLI1 type I fusion transcript while 6 patients EWS-FLI1 type II; in 7 samples we couldn’t find any fusion transcript although their RNA was good. We tested 5 of these negative samples with Real-Time PCR and we found 2 patients who were carriers of EWS-FLI1 type I fusion transcript. CD99 resulted positive in 34 samples out of 54.

The detection of fusion transcripts using RT-PCR methods can be useful as a support to EFT diagnosis. Moreover the possibility to assess a Real-Time PCR assay enhances analysis sensibility and minimizes the chance of false positives. EFT cytogenetic characterization completes morphologic and immunophenotipic data allowing a more careful classification and an identification of subgroups with different prognosis.

Epithelioid hemangioendothelioma (EHE) is a rare vaso-formative tumor of variable biological behavior that has been considered a tumor of borderline malignancy and low-grade angiosarcoma. The majority of cases are associated with low mortality, but some metastasize and cause patient death. Its principal sites of occurrence are soft tissues, liver, lung, and bone. EHE develops as a solitary, painful mass in superficial or deep soft tissue of the extremities and it generally arises from a vessel. Cytogenetic findings are very limited and comprises three reports on totally 4 cases, describing translocations between chromosomes 1 and 3 in two cases, chromosomes 7 and 22 in one case and chromosome 10 and 14 in the last case.

We characterized immunohistochemically 5 cases of this tumour type and currently we are performing Real-Time PCR assays to analyze the expression of two genes (MDM2 and CDK4) known to be involved in pathogenesis of tumours.

Three out 5 patients presented epithelioid hemangioendothelioma of the bone while two affected soft tissues. All the samples showed positivity for CD34 and CD31; 4 samples out 5 were also positive for FLI1. We tested Factor VIII immunostaining on 3 of these cases which resulted positive; EMA was positive on 3 samples out 5. Cytocheratins (AE1/AE3, CAM 5.2 and CK7) were always negative except in one case which showed CAM 5.2 positivity. Our preliminary results by Real-Time PCR analysis performed on 5 cases suggest that MDM2 and CDK4 have a different expression in epithelioid hemangioendotheliomas compared to normal tissue.

Our study shows that use of molecular techniques such as Real-Time PCR could complement histopathological diagnosis in order to identify a marker of biologic behaviour of this enigmatic tumour type.

Synovial sarcomas are mesenchimal tumours with unde-fined histogenesis which represent 5–10% of soft tissues tumours; they are divided into different subtypes according to morphology and epithelial differentiation. From a molecular point of view, synovial sarcoma is characterized by t(X;18)(p11;q11) translocation which joins SYT gene with a member of SSX gene family. We developed an efficient method to detect the two main fusion transcripts SYT-SSX1 and SYT-SSX2 based on RT-PCR or Real-Time PCR applied to archival paraffine-embedded samples.

This study includes 49 patients surgically treated for synovial sarcoma and analyzed with routine immuno-histochemical analysis. We used alternatively nested-PCR or Real-Time PCR, with SYBR green method, to detect SYT-SSX transcripts: these techniques allowed us to discriminate between the two transcripts.

In 42 subjects out of 49 we could find a specific fusion transcript and, in particular, 31 patients were carriers of SYT-SSX1 translocation. Interestingly we could find 6 patients who were carriers of both SYT-SSX1 and SYT-SSX2 transcripts. We selected nine samples for Real-Time PCR analysis and we could quantify the reciprocal ratio between the two fusion transcripts when they were both present in the same sample.

The use of molecular techniques such as RT-PCR represents a sensitive and reliable tool as a support to histopathologic diagnosis of synovial sarcoma. Moreover, Real-Time PCR enormously enhances sensibility and enables to determine single transcript quantity when both SYT-SSX1 and SYT-SSX2 are present in the same sample. This method can also be used to reclassify those cases whose diagnosis is still undefined after routine analysis.