10 Minutes Talk

吳 華 席Hua-Hsi Wu, MD

OB/GYN, VGH-TPEAug 12, 2008

Impact of Determining PALN Metastases/Gold et al

Ovarian cancer, MicroRNA, Exosome

Impact of Determining PALN Metastases/Gold et al

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?

Impact of Determining PALN Metastases/Gold et al

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 & ce

ll death

Impact of Determining PALN Metastases/Gold et al

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

Impact of Determining PALN Metastases/Gold et al

Biogenesis of microRNAs

Impact of Determining PALN Metastases/Gold et al

Detailed structures of pri-, pre- and mature miRNAs

Impact of Determining PALN Metastases/Gold et al

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)

Impact of Determining PALN Metastases/Gold et al

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.

Impact of Determining PALN Metastases/Gold et al

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

Impact of Determining PALN Metastases/Gold et al

Results

Impact of Determining PALN Metastases/Gold et al

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.

Impact of Determining PALN Metastases/Gold et al

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

Impact of Determining PALN Metastases/Gold et al

**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

Impact of Determining PALN Metastases/Gold et al

Comparison of miRNA with peripheral blood-derive

d exosomes and their corresponding tumors

Impact of Determining PALN Metastases/Gold et al

miRNAs in exosomescompared by stages

Impact of Determining PALN Metastases/Gold et al

Comparison of specific exosomal miRNAs

immediately after blood draw or 24, 48, & 96 hr later

Impact of Determining PALN Metastases/Gold et al

Appendix A

Impact of Determining PALN Metastases/Gold et al

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.

Impact of Determining PALN Metastases/Gold et al

Conclusions

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