Valuation Of PAX3-FKHR/PAX7-FKHR And MiRNA-1 In The Classification And Prognosis Of Orbital Rhabdomyosarcoma

Rhabdomyosarcoma(RMS) is the most common primary malignant tumor in childhood,accounting for 5%of all malignant tumors and 50%of all soft tissue sarcomas in children.RMA originates from mesoblastic undifferentiated multipotent mesenchymal stem cells,characterized by high malignancy,fast progression and high mortality, seriously endangering patient lives.Orbital RMS(ORMS) accounts for 12%of all RMSs,and 25-35%of head and neck RMSs,the incidence of which ranks the first in orbital malignant tumors.The prognosis of ORMS varies greatly with multiple factors.At present the diagnosis of ORMS mainly depends on general pathology and immunohistochemistry.Pathologically,ORMS is classified as embryonal rhabdomyosarcoma(ERMS),alveolar rhabdomyosarcoma (ARMS) and pleomorphic rhabdomyosarcoma(PRMS),of which ERMS is the most common with relatively good prognosis.ARMS and PRMS are rare,but they are more pernicious with relatively poor prognosis.As the prognosis and treatment of these subtypes of RMS are different,accurate diagnosis and classification are of primary importance.However,current pathologic and immunohistochemical techniques presently available are mostly helpless for the diagnosis and classification of some RMS cases. There is a need to seek new diagnostic means.Advances in molecular biology make the research in this field possible.The present study was attempted to explore the scientific feasibility of using fusion gene PAX3-FKHR,PAX7-FKHR and miRNA as a supplementary means of pathologic diagnosis,and investigate its relationship with prognosis.PAX3-FKHR and PAX7-FKHR in RMS are induced by translocation of chromosome t (2;13) and(q35;q14) and formed by connection between the DNA binding domain of myogenic transcription factor PAX3 5' end and the transactivation domain of another transcription factor FKHR 3' end.PAX3-FKHR and PAX7-FKHR participate in multiple metabolisms,affecting tumor development and progression.By detecting the existence of the above fusion genes,tumor classification and prognostic assessment could be anticipated.miRNA is a resolution conservative,non encoded and single-chained small RNA family containing 18-25 nucleotide bases.It plays important roles in cell physiopathologic processes by regulating expressions of different target genes.miRNA is involved in organ genesis,tissue differentiation,cell proliferation,lipid metabolism, apoptosis and tumor occurrence.It is reported in the literature that miRNA can also be used for tumor grading.There has been no study reporting miRNA expression in RMS. The present study was attempted to explore the potential use of miRNA in the assessment of tumor prognosis by detecting its expression in RMS.Clinical data of 29 patients with ORMS who received surgical treatment in the Institute of Orbital Diseases of the General Hospital of the Chinese Armed Police Force between March 2001 and February 2008 were analyzed retrospectively.All the patients were followed up.The pathologic specimens of the 29 ORMS cases were HE stained routinely and observed histopathologically.All the specimens were immunohistochemically stained with respect to Vimentin,MyoD1,Myoglobin,actin,Desmin,CD99,Ki-67 and P53 antigens.Ten cases of non ORMS small round cell tumors were used as pathologic controls,and 5 cases of normal striated muscle(from cosmetic surgery) were used as normal controls.Using total RNA extracted from the paraffin-embedded tumor tissues as the template and P-actin as the internal reference,RT-PCR and semi-nested PCR were performed to detect PAX3-FKHR and PAX7-FKHR.The PCR product was confirmed by DNA sequencing.miRNA expression in ORMS of 13 pathologic specimens and 5 control specimens was detected by microarrays containing 1,320 probes,and the results were analyzed by microarray difference analysis software.Specific expression of miRNA was verified by real time PCR.Statistical treatment was conducted by SPSS 13.0 software. Survivals were compared by Kaplan-meiler method.Of the 29 ORMS cases,19 had typical morphologic presentation and immunohistochemical staining,and were diagnosed as ERMS histopathologically;2 cases had characteristic fibrovascular septa and alveolar-like structures,and were diagnosed as ARMS;and the remaining 8 cases had no typical presentation,with poorly differentiated tumor cells.According to immunohistochemical staining,they were grossly diagnosed as ORMS,without further grading.RNA was extracted from 27 of the 29 cases.Expression of PAX3-FKHR and PAX7-FKHR was detected in 7 cases.No expression of PAX3-FKHR and PAX7-FKHR was detected in the pathologic and normal control groups.The results of microarray detection revealed that expression of hsa-miR-106a, hsa-miR-18b and hsa-miR-92a was increased markedly in tumor tissues,the highest being 11.8 fold than the normal control groups.While expression of hsa-miR-29a, hsa-miR-22,hsa-miR-133b,hsa-miR-133a,hsa-miR-29b,hsa-miR-1 and hsa-miR-29c was decreased markedly.Real time PCR confirmed that hsa-miR-1 expression in tumor tissue was decreased markedly.Taking the follow-up data into account,the extent of decrease in expression of these genes was correlated with the prognosis of the patients: the greater the extent of decrease the poorer the prognosis.It is concluded that general pathology and immunohistochemistry are helpful in diagnosis and classification of most typical ORMS cases,but seem difficult for some poorly differentiated atypical cases.PAX3-FKHR/PAX7-FKHR and miRNAl can be used as effective reference markers for diagnosis and classification of ORMS,as well as for judgment of prognosis.In addition,the present study first found differentiated expression of 145 miRNAs in ORMS,of which 10 were most prominent.This finding provides a new way of research thinking.It is hoped that this preliminary study about the role of PAX3-FKHR/PAX7-FKHR and miRNAl in the pathogenesis of ORMS would provide some experimental clues for further study of pathogenesis of ORMS and gene therapy in future.