Relationship Of MyoD1 And Myogenin Expression In Rhabdomyosarcomas To DNA Ploidy Imbalances And Identification Of PAX3-FKHR Fusion Gene

OBJECTIVE: To investigate application of myogenic transcriptional regulatory proteins in diagnosis and differential diagnosis of Rhabdomyosarcomas. To explore clinical value of the florescent in situ hybridization (FISH) technique for identification of PAX3-FKHR fusion gene in paraffin wax embedded rhabdomyosarcomas tissues. To correlate myogenic transcriptional regulatory proteins & DNA ploidy in rhabdomyosarcoma with other prognostic factors and patient survival to test their possible prognostic value for rhabdomyosarcomas. MATERIAL and METHODS: paraffin wax embedded rhabdomyosarcomas tissues of 128 cases were conserved by Tianjin Cancer Hospital during 1983-2003. There was a control group with 30 cases of non- rhabdomyosarcomas (peripheral neuroectodermal tumor, leiomyosarcoma, synovial sarcoma, non-Hodgkin lymphoma, malignant fibrous histiocytoma for every 6 cases) in the first part. The methods including Tissue Microarray Power VisionTM Two-Step Immunohistostaining, Florescent in situ hybridization (FISH) and Flow cytometry. We used the chi 2 test to correlate DNA ploidy with other prognostic factors, Kaplan-Meier procedure to estimate overall survival in terms of individual prognostic factors, log-rank test to calculate differences in survival between groups and Cox multivariate regression analysis to determine the independence of variables in relation to survival.RESULT: Immunohistochemical found MyoDl and myogenin stained positively in rhabdomyosarcomas cellular nucleus for 76. 56% (98/128) and 81. 25% (104/128) , Both of them could not be detected in normal striated muscle; a -Sarcometic Actin, myoglobin, myosin, Desmin, Vimentin NSE all stained positively in cytoplasm , The positive rat of a -SarcometicActin, myoglobin, myosin, Desmin-. was distinguished 57. 03%, 48. 43%, 46. 09%, 69. 53%, 84. 38% and 10. 94%. The expression sensibility of these factors ranked from high to low was Vimentin, myogenin, MyoD1, Desmin a-Sarcometic Actin, myoglobin myosin and NSE; The expression specificity of these factors ranked from high to low was MyoDK myogenin, NSE, myoglobin, myosin, Vimentin and Desmin; The experiment effective power of these factors ranked from high to low was myogenin, MyoD1, Vimentin, Desmin, a -Sarcometic Actin, myoglobin, myosin and NSE. Our data show that staining for MyoDl and myogenin will be a simple, rapid, and accurate adjunctfor distinguishing between rhabdomyosarcomas and non- rhabdomyosarcomas. Florescent in situ hybridization (FISH) result: These single-copyFISH (scFISH) probes of our study were designed by computational sequence analysis . There were only 7 cases in the group that we had detected the fusion gene of PAX3-FKHR. All of these cases were ARMS with higher grade than II .MyoD1 and myogenin were strong expressed more than III grade.Exept one missed, all the other 6 pateints were dead.Flow cytometry result: A statistically significant correlation was found between DNA ploidy histologic subtype and MyoDl&myogenin of RMS,. A hyperdiploid DNA pattern predominated among patients with embryonal RMS, and a tetraploid pattern dominated among patients with alveolar RMS. The highest 5-year survival rate was seen among patients with hyperdiploid RMS, followed by those with diploid, tetraploid and hypertetraploid RMS. Although DNA ploidy was a significant prognostic factor in univariate analysis, it did not retain its independent prognostic value in multivariate analysis, in which histologic subtype, histologic grdae , stain grade of MyoDl & myogenin, P53 positive expression and CD31 positive expression were the only significant factors.CONCLUSION: The immunohistochemical staining of paediatric rhabdomyosarcomas with antibodies to MyoDl and myogenin provides sensitive and specific diagnostic information. Almost all cases demonstrate nuclear expression of both products, but myogenin immunostaining is usually more clinically useful because it is more consistent and is associated with less non-specific staining. The sequence of the probes that we designed for Floresce...