Purpose: To investigate the link between microsatellite instability and epigenetic silencing of the MLH1 gene in the human retinoblastoma genome.
Methods: Methylation at the 5' region of MLH1 was studied, along with its protein expression level by using immunohistochemical staining in 51 retinoblastoma tumors and 2 retinoblastoma cell lines. Also assessed was the genomic stability of 26 retinoblastoma DNAs from microdissected tumor tissue and matched normal retina tissue obtained from the same patient by microsatellite instability (MSI) analysis. The National Cancer Institute-designed reference panel, and 85 markers on chromosomes 1, 6, 9, and 13 were used.
Results: Hypermethylation of the MLH1 promoter was detected in the WERI-Rb1 cell line and in 34 (67%) of the 51 tumors, but not in cell line Y79 and the other 17 tumors. MLH1 hypermethylation was associated with null MLH1 protein expression (P < 0.0005) and with well-differentiated histology (P < 0.05). MSI at three markers (D2S123, D6S470, and D13S265) was frequently identified among 26 retinoblastoma specimens with matched normal DNA. Among these 26 retinoblastomas, high-frequency MSI (MSI-H) tumors were detected in 19% (5/26) and low-frequency MSI (MSI-L) in another 19% (5/26). The remaining 62% (15/26) were genetically stable (MSS). MSI status (MSS, MSI-L, and MSI-H) was not associated with MLH1 promoter hypermethylation (P = 0.088; Kruskal-Wallis test).
Conclusions: Epigenetic silencing of the DNA repair gene MLH1 by promoter hypermethylation is a frequent event in retinoblastoma. The results showed that somatic genetic changes involving MSI occur in a subset of retinoblastoma and implicated the presence of a defective DNA mismatch repair pathway resulting in MSI in retinoblastoma.