The Study Of Fusion Gene And Histogenesis On Clear Cell Sarcoma Of Soft Tissue

The expressions of S-100 and HMB-45 determined with the immunohisto-chemical method is the main way to the diagnosis and differential diagnosis of clear cell sarcoma.And there is no other specific and sensitive marker to diagnosis it,especially differential diagnosis with atypia cases.The translocation t(12,22)(q13,q12)leads to the fusion of EWS and ATF1 gene.The t(12,22)(q13,q12)and EWS-ATF1 fusion gene are molecular genetics features.In addition,detecting EWS-ATF1 fusion gene and finding the different breakpoint are useful for the diagnosis and prognosis of clear cell sarcoma.The histogenesis of this tumor has long been controversial.In the present,2002 WHO classification of soft tissue tumors,clear cell sarcoma are incuded in the group of uncertain classification malignant neoplasms.Deriving from synovial membrane or neural crest are the two ideas of histogenesis existed for some years.To assess the feasibility of detection of EWS-ATF1 fusion gene and to investigate the diagnostic significance of detecting chimeric mRNA in paraffin-embedded tumor tissues of clear cell sarcoma(CCS).To analyze the relations between different breakpoints of fusion gene and clinical prognosis of clear cell sarcoma.And to discuss the genesis of CCS by detecting the expressions of MITF mRNA and MITF protein with RT-PCR and immunohisto-chemical method.From 1992 to 2006,26 cases of formalin-fixed,paraffin-embedded tumor tissues of CCS,5 cases of fresh frozen tissues and 20 cases of controls(6 cases of Ewing's sarcomas,5cases of PNETs,2 cases of alveolar soft part sarcomas,2cases of malignant melanomas,5cases of synovial sarcomas)were collected.HE stain sections are available for all examples.And all the cases have the final diagnosises determined by above two pathologists.The expressions of S-100 and HMB-45 were determined with the SP immunohisto-chemical method in 26 cases of formalin-fixed, paraffin-embedded tumor tissues of CCS.And the expression of MITF was determined with the SP method in 24 cases chosen from 26 cases of formalin-fixed, paraffin-embedded tumor tissues of CCS.Ultrastructural examination was performed on 5 cases of fresh frozen tissues.RNA was extracted from paraffin-embedded tissue, and EWS-ATF1 mRNA expression was detected by RT-PCR and nested-PCR.β-actin,β_2-microglobulin and GAPDH were used as internal control. MITF transcripts of 5 cases of fresh frozen tissues were detected by RT-PCR.Products were routinely sequenced.Ultrastructural examination was performed on 5 tumors.Representative fresh tumor tissue was fixed in 2%glutaraldehyde,postfixed in 1%osmium tetroxide,and embedded in epoxy resin using standard procedures.Results:β-actin andβ_2-microglobulin were detected in 22/26 and 24/26 clear cell sarcoma,respectively.EWS-ATF1 fusion transcripts were detected in 20 of the 24 CCS cases(16 cases typeⅠ,0 cases typeⅡ,4 cases typeⅢ).The remaining 4 cases were negative for EWS-ATF1.No EWS-ATF1 mRNA expression was detected in all the controls.The general expression rate of S-100 and HMB-45 was 79.2%,70.8%, respectively.RT-PCR analysis for the GAPDH was detected in 4 of 5 cases tested. Melanocyte-specific splice form of the MITF transcript was positive in 4 of 4 cases (4/4).Most cases showed immunostaining for MITF(20/24).Electron Microscopic Findings:ultrastructural appearance of most cases with melanosomes in different stages,and the presence of glycogen,swollen mitochondria and basilar membrane.Conclusion:detection of EWS-ATF1 fusion gene from formalin-fixed paraffin-embedded materials by nested RT-PCR and RT-PCR is feasible.EWS-ATF1 detection can be used as a highly assistant diagnostic tool for the diagnosis and differential diagnosis of CCS.CCS expresses the melanocyte-specific form of the MITF transcript,further supporting its genuine melanocytic differentiation theory to some extent.We can deduce that clear cell sarcoma is a special malignant tumor which derived from neural crest and be available in tendons and aponeuroses.