Evaluating RASSF1 promoter methylation and the 16-gene signature as a prognostic marker in hepatoblastoma.

Abstract

63 Background: Hepatoblastoma (HB) is the most common malignant liver tumor in childhood. Although combination of neoadjuvant chemotherapy and surgery has improved the outcome of standard-risk HB-patients up to 94% the outcome of high-risk HB-patients with metastatic tumors or invasion of large hepatic veins is still dismal and only 60% of these patients survive. Therefore, the identification of biomarkers that could help early detection of high-risk HB-patients and lead to risk-adapted therapies is of utmost importance. Methods: Genomic DNA of tumor and normal liver samples from 21 HB patients was extracted with standard procedures and bisulfite-treated using EpiTect Bisulfite Kit (Qiagen). Methylation status of the Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) gene was analyzed by methylation-specific-PCR. cDNA of tumor and normal liver samples was used to determine the 16-gene expression signature according to Cairo et al. (2008, Cancer Cell) using real-time PCR. Association studies of molecular and clinical data were performed with GraphPad Prism Version 3.0 using two-tailed Chi square test. Results: We identified 9/21 HB cases hypermethylated in the RASSF1 promoter region. Interestingly, RASSF1 promoter methylation was significantly associated with metastases (p=0.0166) and occurrence of this epigenetic marker was associated with a reduced overall survival of patients (p=0.0008). Moreover, we discriminated HB tumors in C1 and C2 subclasses by using the 16-gene signature and correlation of these subtypes with the methylation status of RASSF1 showed a significant association (p=0.0037) for aggressive C2 tumors (8/11), but not C1 tumors (1/10). Conclusions: RASSF1 promoter hypermethylation is a strong prognostic marker for metastasis and worse outcome and correlates with an aggressive C2 HB-tumor profile. Since the detection of tumor-derived methylated DNA in the blood stream of cancer patients is nowadays practical, our data advise RASSF1 methylation assays to be used for risk assessment in HB-patients in the future.