(A) Methylation status of the IGFBP3 promoter in pediatric liver cancers. Summary of the analyses of the IGFBP3 methylation status in the 36 hepatoblastomas (HB), 9 pediatric hepatocellular carcinomas (HCC), and 7 normal pediatric liver samples (NL), as determined by MSP. The black boxes represent the detection of methylated alleles, and the white boxes depict unmethylated alleles. (B) Methylation-specific PCR (MSP) of pediatric liver cancers. The promoter region of the IGFBP3 gene was analyzed for methylated CpG sites by MSP using bisulfite-treated genomic DNA from pediatric liver tumors (T) and normal liver tissue (N). Representative images of the MSP experiments are shown, revealing PCR products for unmethylated (U) and methylated alleles (M). In vitro methylated DNA (SssI) was used as a positive control for methylated alleles. (C) Overall survival (OS) stratified by methylation status. OS was calculated as the time from diagnosis to death from disease and is plotted for 8 HB patients with (orange line) and 25 without (black line) IGFBP3 promoter methylation over a period of 100 months. Statistical significance was calculated using Mantel-Cox test.