Detection of promoter hypermethylation of RARβ, RASSF1A, DAPK and P16/ INK4a genes in cell-free DNA from serum (s) and pleural liquid (pl) of patients (p) with pleural effusion

Abstract

9637 Background: Aberrant gene promoter methylation (AM) is a major mechanism for tumour suppressor gene (TSG) silencing in all tumour types and affects their development and aggressiveness.AM of RARβ,RASSF1A, DAPK,P16/INK4a genes have shown to be involved in most tumors. Objectives: 1) to assess the concordance of gene promoter hypermethylation in paired s and pl samples of cancer p with malignant pleural effusion 2) to investigate their role as a marker of pleural effusion malignancy. Methods: We evaluated 53 consecutive p presenting pleural effusion and malignancy(24 lung,11 digestive,8 breast,4 ovary,6 others) at the General Hospital of Alicante (Spain).Hypermethylation of TSG were investigated by using methylation-specific PCR(MSP)after sodium bisulphite treatment. Results: Conventional cytology of pleural effusion samples revealed neoplastic cells in 17 of 48 p (35,5%). AM of at least one of the genes tested was detected in 24 out of 53 p (45,3%) in s and in 31 out of 53 p (58,6%) in pl. In pl 34% had only 1 gene methylated,17, 0% had 2 genes methylated, 5,7% had 3 genes methylated and only 1,9% had 4 genes methylated. In s,14% had only 1 gene methylated,8% had 2 genes methylated and only 2% had 3 genes methylated. Concordance in the pattern of aberrant methylation was found in paired s and pl samples of 13 p. In 11 p, methylation was found only in pl and not in s; only in 3 patients was found in serum and not in pleural liquid. AM of DAPK was found in 7 p (13,2%) in s and 10 p (18,9%) in pl; in 10 p (18,9%) in s and 20 p (37,7%) in pl for P16/INK4a; in 9 p (17%) in s and 8 p (15,1%) in pl for RASSF1A and in 10 p (18,9%) in s and 11 p (20,8%) in pl for RARβ. Correlation coefficient between s and pl methylated genes was r=0,6 p<,0001. Median survival time of p with at least one gene methylated in pl was 12,63±5,95 mo; for p with none of genes methylated median survival has not yet reached. Conclusions: 1) Aberrant gene promoter methylation of TSG can be detected in pleural liquid 2) In pleural liquid, 58,6% of p were found to have at least one TSG methylated vs 35,5% of p with neoplastic cells by conventional cytology 3) Aberrant methylation showed a negative prognostic effect. No significant financial relationships to disclose.