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10 Minutes Talk

10 Minutes Talk. 吳 華 席 Hua-Hsi Wu, MD OB/GYN, VGH-TPE Aug 12, 2008. Ovarian cancer, MicroRNA, Exosome. Introduction Ovarian cancer. Worldwide, 6th most common in female & 125,000 deaths annually 75% diagnosed as extra-ovarian dz (EM ca 73%, Breast ca 55% & Cx ca 50% in stage 1)

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10 Minutes Talk

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  1. 10 Minutes Talk 吳 華 席 Hua-Hsi Wu, MD OB/GYN, VGH-TPE Aug 12, 2008

  2. Ovarian cancer, MicroRNA, Exosome

  3. IntroductionOvarian cancer • Worldwide, 6th most common in female & 125,000 deaths annually • 75% diagnosed as extra-ovarian dz (EM ca 73%, Breast ca 55% & Cx ca 50% in stage 1) • 5-yr survival: stage 1 > 90%, advanced stage 21% • How to early Dx?

  4. IntroductionMicroRNA • Small (21-25 nucleotides in length) non-coding RNAs • Suppress the translation of target mRNAs by binding to their 3” untranslateed region • Post-transcriptional silencing • Cleavage of homologus mRNA • Specific inhibition of protein synthesis • Critical regulator of cellular processes • Proliferation, differentiation, development & cell death

  5. IntroductionMicroRNA • Distinct MicroRNAs signature, classify human cancers • Ovarian cancers: • miR-21, miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205 & miR-214 • Up-modulation of specific microRNA could be the amplification of its gene. • Is microRNAsignature better than mRNA signature, in cnacer • Dx, staging, progression, and response to treatment

  6. Biogenesis of microRNAs

  7. Detailed structures of pri-, pre- and mature miRNAs

  8. IntroductionExosome • small (50-100nm) membrane vesicles of endocytic origin, in the peripheral circulation (of ovarian cancer patients was found initially in 1979) • Source • Except cancers, including reticulocytes, dendritic cells, B cells, T cells, mast cells, epithelial cells, and embryonic cells. • All exosomes share certain common characteristics (structures, size, density & general protein composition)

  9. IntroductionExosome • Cell-cell communication • Directly by surface expressed ligands • By transferring molecules between cells • Exosomes contains both cellular mRNA and miRNAs • While tumor-derived exosomes exhibit some common, shared proteins, they also express an array of tumor antigens that reflect the originating tumor cells.

  10. Material and Methods • Serum & biopsied tissue • Serous papillary adenocarcinoma • Stage I n=10, stage II n=10, stage III n=20, stage IV n=10 • Ovarian adenoma n=10 • NED of ovarian diseases n=10 • Isolation of circulating exosomes • MACS (EpCAM, anti-epithelial cell adhesion molecules) • Isolation & profiling of miRNAs • mirVana microRNA isolation kit

  11. Results

  12. Panel A: The levels of circulating tumor-derived exosomes compared to stage of ovarian cancer Panel B: Electron micrograph of circulating exosomes isolated by magnetic beads.

  13. Presence of small RNA associated with circulating EpCAM-positive exosomes from ovarian cancer patients

  14. **Intensities for specific microRNAs derived from the advanced-staged ovarian tumors (□) and from EpCAM-positive exosomes (■) isolated from the sera of these patients. **miR-21,miR-141, miR-200a, miR-200b, miR-200c, miR-203, miR-205, and miR-214 have previously been demonstrated to be upregulated markers for ovarian cancer

  15. Comparison of miRNA with peripheral blood-derived exosomes and their corresponding tumors

  16. miRNAs in exosomescompared by stages

  17. Comparison of specific exosomal miRNAsimmediately after blood draw or 24, 48, & 96 hr later

  18. Appendix A

  19. Results • MicroRNA from ovarian tumor cells and exosomes from the same patients were positive for 218 of 467 mature microRNAs analyzed. • The levels of the 8 specific microRNAs were similar between cellular and exosomal microRNAs (exhibiting correlations from 0.71 to 0.90). While EpCAM-positive exosomes were detectable in both patients with benign ovarian disease and ovarian cancer, exosomal microRNA from ovarian • cancer patients exhibited similar profiles, which were significantly distinct from profiles observed in benign disease. Exosomal microRNA could not be detected in normal controls.

  20. Conclusions • These results suggest that microRNA profiling of circulating tumor exosomes could potentially be used as surrogate diagnosticmarkers for biopsy profiling, extending its utility to screening asymptomatic populations.

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