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S. Masuda
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P2.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 225)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.08-033 - DNA Methylation on Promotor Region of RASSF1 Gene in Thymic Neuroendocrine Tumor Is Higher than B3 Thymoma and Thymic Squamous Cell Carcinoma (ID 2544)
09:30 - 09:30 | Author(s): S. Masuda
- Abstract
Background:
RASSF1 gene, located in 3p21.3, has eight exons and two promotor regions. RASSF1 is very famous tumor suppressor gene in various cancers. It was reported that DNA methylation on promotor region of RASSF1 in lung cancer, bladder cancer, and breast cancer and so on was higher, additionally low expression of RASSF1 was possible to cause to be poor prognosis. It is few reports about epigenome status in thymic epitherial tumors. We planned to explore DNA methylation in thymic epitherial tumors cyclopedically.
Methods:
①DNA and RNA were extracted from frozen specimen of B3 thymomas (8cases), thymic cancers (8cases), and thymic neuroendocrine tumors(NET)(3cases). ②DNA was treated by bisulfite conversion. ③DNA methylation level in 470000 CpG sites were measured by infinium methylation assay (Human methylation 450K; ILLMINA) exhaustively. ④DNA methylation on promotor regions of RASSF1 was measured by pyrosequencing（PyroMARK[TM]system;QIAGEN）. ⑤Expression level of mRNA was measured by Real time RT-PCR(Thermal Cycler Dice® Real Time System Single; Takara), using TaqMan Gene Expression Assays (Hs00200394_m1；Applied Biosystems). Internal reference gene is GAPDH（Hs02758991_g1；Applied Biosystems). ⑥Expression level of protein was analysed by immunostaining. Anti-RASSF1a antibody（Anti-RASSF1a antibody [3F3] ab23950, Mouse monoclonal, abcam）was used by CSAⅡmethod（DAKO CSA II, Biotin-Free Catalyzed Amplification System）.
Results:
Significant difference of DNA methylation was recognized by analysis of infinium methylation assay. All 11 CpG sites were configured on 1α promotor region of RASSF1 in this assay. This assay showed DNA methylation level was highest in NET group. DNA methylation level were 70.9±4.9% in NET, 22.2±20.0% in thymic cancer, 14.3±12.3% in B3 thymoma. ( NET vs Cancer/B3 t-test：P<0.00001). Pyrosequencing showed DNA methylation level were 24.0±13.1% in NET, 3.0±0.5% in thymic cancer, 3.0±0.9% in B3 thymoma. Real time RT-PCR showed that relative expression level (/normal thymus) were 0.48±0.31 in NET, 1.02±0.82 in carcinoma, 2.13±2.93 in B3 thymoma ( NET vs Carcinoma/B3 t-test：P=0.16). Immunostaining of RASSF1 was scored by stain intensity and stain extend. Immunostaining scoring of RASSF1 showed expression inhibition rate were 66% in NET, 50% in thymic cancer, 14% in B3 thymoma.
Conclusion:
The infinium methylation assay showed that DNA methylation on promotor region of RASSF1 in NET is higher than B3 thymoma and thymic cancer. The pyrosequencing validated this result. It was tendency to suppress the mRNA or protein expression of RASSF1 in NET, compared to other tumors. It is possible that aberrant DNA methylation on promotor region of RASSF1 may be specific change in NET among thymic epitherial tumors. Now we collected 8 formalin-fixed paraffin-embedded samples of thymic NETs to perform pyrosequencing and immunostaining of RASSF1 gene.
