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E2F3 in Soft Tissue Sarcoma Development

E2F3 in Soft Tissue Sarcoma Development. M. Scurr; A Feber; J Shipley; A Fletcher; N Dennis; I Judson; C Cooper. Growth Factors. Cyclin D. RB. E2F3. CDK. P. RB. P. P. Active E2F3. G1/S Transition. Cyclin E, CDK2. DNA synthesis (TS,TK,DHFR) Cyclin A. E2F3 Overexpression and Cancer.

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E2F3 in Soft Tissue Sarcoma Development

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  1. E2F3 in Soft Tissue Sarcoma Development M. Scurr; A Feber; J Shipley; A Fletcher; N Dennis; I Judson; C Cooper

  2. Growth Factors Cyclin D RB E2F3 CDK P RB P P Active E2F3 G1/S Transition Cyclin E, CDK2 DNA synthesis (TS,TK,DHFR) Cyclin A

  3. E2F3 Overexpression and Cancer Human Bladder Cancer (Feber et al) E2F3 an oncogene activated by gene amplification and overexpression. E2F3 overexpression correlates with increasing grade and stage Prostate Cancer (Foster et al) E2F3 overexpression an independent variable overall survival (P=.0014) cause specific survival (P=.0004)

  4. Evaluation of Potential Role for E2F3 in STS RB1 mutation/loss in STS ~1/3 of leiomyosarcomas Up to 70% of primary tumours altered RB expression Reports of cyclin D1/Cdk4 overexpression Implicating RB-E2F Axis

  5. STS Tissue Microarrays 165 patients with primary tumour samples and clinical data

  6.  Fibrosarcoma Leiomyosarcoma   MFH Monophasic Synovial Sarcoma

  7.  Epithelial cell staining Spindle cell staining

  8. E2F3 Nuclear Staining:Total STS TMA

  9. Leiomyosarcoma Fibrosarcoma MFH Synovial Sarcoma

  10. Prostate Cancer Synovial Sarcoma

  11. Clinical Correlations: E2F3 Staining and Prognostic Variables. • Size • Stage • Grade

  12. Clinical Correlations: E2F3 Staining and Prognostic Variables. • Size No significant correlation • Stage No significant correlation • Grade

  13. Clinical Correlations: E2F3 Staining and Prognostic Variables. • Size No significant correlation • Stage No significant correlation • GradeP<0.001

  14. Clinical Outcomes: Disease Free Survival UNIVARIATE ANALYSIS size stage grade MULTIVARIATE ANALYSIS stage +ve E2F3 nuclear staining HR 1.41 (95% CI .89-2.44) P=0.148

  15. Clinical Outcomes: Disease Free Survival UNIVARIATE ANALYSIS size stage grade MULTIVARIATE ANALYSIS stage +ve E2F3 nuclear staining HR 1.45 (95% CI 0.93-2.25) P=0.1

  16. Clinical Outcomes: Cause Specific Survival UNIVARIATE and MULTIVARIATE ANALYSES size stage grade site

  17. Clinical Outcomes: Cause Specific Survival UNIVARIATE and MULTIVARIATE ANALYSES size stage grade site +ve E2F3 nuclear staining HR 1.57 (95% CI 0.92-2.7) P=0.10

  18. Assessment of biological function of E2F3 overexpression in STS

  19. HT1080 HTB-88 HTB-114 SKW SS-SY-II BAX FUJI 5637 (+ve control) E2F3 in STS Cell Lines E2F3 GAPDH

  20. HT1080 HTB-88 HTB-114 SKW SS-SY-II BAX FUJI 5637 (+ve control) E2F3 in STS Cell Lines E2F3 GAPDH

  21. Si-1 Si-2 Si-3 Si-all Scr-1 Scr-2 Scr-3 Scr-all control E2F3 siRNA Transfection: 24 hours  E2F3 GAPDH

  22. Biological effect of “knocking down” E2F3 production Si-3 and Scr-3 transfection of HT 1080 Analysis of effect on G1/S transition BrdU Incorporation: Immunofluorescence Microscopy fluorescent activated cell sorting (FACS)

  23. BrdU Incorporation Immunofluorescence Microscopy Scr-3 Si-3

  24. BrdU Incorporation: FACS Si-3: 0 hrs Si-3: 24 hrs post Scr-3: 0 hrs Scr-3: 24 hrs post

  25. Conclusions • 62% STS samples positive E2F3 nuclear staining • LMS/MFH and fibrosarcoma predominantly <20% intensity • Synovial sarcoma more intense staining similar pattern to prostate and bladder cancer. • +ve Nuclear staining correlates with higher grade • Non-significant trend to poorer disease free and cause specific survivals • E2F3 knockdown studies demonstrate that inhibition of E2F3 expression inhibits cell cycle progression. E2F3 OVEREXPRESSION HAS A ROLE IN THE MALIGNANT PHENOTYPE IN STS

  26. Institute of Cancer Research Molecular Carcinogenesis Colin Cooper Andy Feber Sandra Edwards Penny Flohr Toby Roe Jeremy Clark Janet Shipley Anne Fletcher Nening Dennis Cancer Therapeutics Jenny Titley Mike Walton Royal Marsden Hospital Sarcoma Unit Ian Judson Omar Al-Muderis Histopathology Department Cyril Fisher Statistics Roger Ahern University of Liverpool Department of Pathology and Medical Genetics Chris Foster Andy Dodson Acknowledgements Supported by: Royal Marsden NHS Trust Charitable Fund Grant Jeannette Pomeraniec Sarcoma Research Fund

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