Despite the progress of therapy, outcomes of advanced hepatoblastoma patients who are refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the mortality rate, novel prognostic markers are needed for better therapy planning. We examined the methylation status of 13 candidate tumor suppressor genes in 20 hepatoblastoma tumors by conventional methylation-specific PCR (MSP) and found hypermethylation in 3 of the 13 genes. We analyzed the methylation status of these 3 genes (RASSF1A, SOCS1 and CASP8) in 97 tumors and found hypermethylation in 30.9, 33.0 and 15.5%, respectively. Univariate analysis showed that only the methylation status of RASSF1A but not the other 2 genes predicted the outcome, and multivariate analysis showed a weak contribution of RASSF1A methylation to overall survival. Using quantitative MSP, we found RASSF1A methylation in 44.3% of the 97 tumors. CTNNB1 mutation was detected in 67.0% of the 97 tumors. While univariate analysis demonstrated RASSF1A methylation, CTNNB1 mutation and other clinicopathological variables as prognostic factors, multivariate analysis identified RASSF1A methylation (p 5 0.043; relative risk 9.39) and the disease stage (p 5 0.002; relative risk 7.67) but not CTNNB1 mutation as independent prognostic factors. In survival analysis of 33 patients in stage 3B or 4, patients with unmethylated tumor had better overall survival than those with methylated tumor (p 5 0.035). RASSF1A methylation may be a promising moleculargenetic marker to predict the treatment outcome and may be used to stratify patients when clinical trials are carried out. ' 2008 Wiley-Liss, Inc.Key words: RASSF1A; CTNNB1; quantitative MSP; hepatoblastoma; prognostic factor Hepatoblastoma is a rare malignant neoplasm of the liver, with an incidence of 0.5-1.5 per million children. 1 Remarkable progress in clinical outcome has been achieved in the past 20 years due to advances in chemotherapy and surgical procedures; however, the mortality rate remains 20-30% and treatment results in patients in advanced stages who are refractory to standard preoperative chemotherapy regimens are unsatisfactory. 2,3 To improve the mortality of these patients, innovative treatment and potent prognostic markers for better therapy planning are needed. The present clinical factors predicting outcome include the level of alpha-feto protein, histology, disease stage and growth pattern of the tumor. 2-4 Chromosomal gains of 2q, 8q and 20 and high expression of telomerase or PLK1 were shown to be moleculargenetic markers predicting poor outcome 5-8 ; however, none have been proven to be independent prognostic factors by multivariate analysis.We previously reported that RASSF1A (RAS association domain family protein 1) methylation, found in 39% of 39 hepatoblastoma tumors, was correlated with poor outcome by univariate analysis. 9 Nevertheless, the article had some limitations that the number of tumors was not enough, the method used to detect the hypermethylation was suboptimal, and the prognostic signifi...
The WT1 gene essential for the embryonic kidney development is mutated in 15-25% of Wilms tumors (WTs). To clarify whether genetic subtypes of WT1 abnormalities are correlated with IGF2 or CTNNB1 alterations or clinicopathological characteristics, we performed comprehensive WT1, IGF2, and CTNNB1 analyses of 36 WTs with WT1 abnormalities using single nucleotide polymorphism arrays, and methylation analysis of the IGF2-H19 differentially methylated region. The tumors were classified into three subtypes based on WT1 abnormalities: 13 with WT1 deletion, 12 with WT1 mutation, and 11 with both deletion and mutation. IGF2 alterations were found in 50% (18/36), paternal uniparental disomy (UPD) of 11p13-11p15 in 13 tumors, UPD limited to 11p15 in 3, and loss of IGF2 imprinting in 2. Quantitative RT-PCR analysis showed that tumors with IGF2 alteration had higher levels of IGF2 mRNA than tumors without IGF2 alteration (P = 0.02). WT1 mRNA levels were very low in six of eight WTs with WT1 deletion, whereas four of eight WTs with WT1 mutation or both deletion and mutation showed higher levels of WT1 mRNA than fetal kidneys. WTs with WT1 mutations occurred in younger patients (P < 0.01), and WTs with mutations or both deletion and mutation (12/23) were more frequent in syndromic patients than WTs (1/13) with the deletion (P = 0.02). WTs with WT1 mutations or both deletion and mutation had the triphasic histological-type (15/23; P = 0.03) and CTNNB1 mutation (17/23; P = 0.03) more frequently than WTs with the deletion (2/13 and 4/13). Thus, three WT1 subtypes were correlated with certain genetic and clinicopathological characteristics.
We evaluated the WT1 and IGF2 status and performed chromosome and/or comparative genomic hybridization analysis in 43 tumor samples from patients with Wilms tumor. On this basis, we classified them into 4 groups: WT1 abnormality, loss of heterozygosity (LOH) of IGF2, loss of imprinting (LOI) of IGF2, and retention of imprinting (ROI) of IGF2, which were seen in 12%, 30%, 16%, and 42% of the tumors, respectively. Patients in the LOI group were older than those in other groups (P < 0.01), and tumors in the WT1 group had fewer cytogenetic changes than did those in the other groups (P < 0.01). It was found that 11q- and +12 were more frequent in the LOI group than in the WT1+LOH+ROI group (P < 0.01 and P < 0.01). There was no difference in the incidence of 16q- between the LOI group and the other groups; however, when we excluded 16 tumors with LOH on 11p15, 16q- tended to be more frequent in the LOI group than in the WT1+ROI group (P = 0.06). The association of 11q- or +12 with LOI of IGF2 found in the present study suggests that many tumors with no WT1 abnormalities need overexpression of IGF2 together with biallelic inactivation of the tumor-suppressor gene on 11q and/or overexpression of growth-promoting genes on chromosome 12. The 11q gene may code for one of the proteins that constitute a CTCF insulator complex, and its mutation, deletion, or haploinsufficiency may cause insulator abnormalities that might lead to LOI of IGF2.
RASSF1A methylation may be a novel molecular-genetic marker for treatment outcome in hepatoblastoma if confirmed by studies examining a larger number of hepatoblastomas.
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