To identify underlying mechanisms involved with metastasis formation in Wilms tumors (WTs), we performed comprehensive DNA methylation and gene expression analyses of matched normal kidney (NK), WT blastemal component, and metastatic tissues (MT) from patients treated under SIOP 2001 protocol. A linear Bayesian framework model identified 497 differentially methylated positions (DMPs) between groups that discriminated NK from WT, but MT samples were divided in two groups. Accordingly, methylation variance grouped NK and three MT samples tightly together and all WT with four MT samples that showed high variability. WT were hypomethylated compared to NK, and MT had a hypermethylated pattern compared to both groups. The methylation patterns were in agreement with methylases and demethylases expression. Methylation data pointed to the existence of two groups of metastases. While hierarchical clustering analysis based on the expression of all 2569 differentially expressed genes (DEGs) discriminated WT and MT from all NK samples, the hierarchical clustering based on the expression of 44 genes with a differentially methylated region (DMR) located in their promoter region revealed two groups: one containing all NKs and three MTs and one containing all WT and four MTs. Methylation changes might be controlling expression of genes associated with WT progression. The 44 genes are candidates to be further explored as a signature for metastasis formation in WT.
BackgroundWilms tumor (WT) is a curable pediatric renal malignancy, but there is a need for new molecular biomarkers to improve relapse risk-directed therapy. Somatic alterations occur at relatively low frequencies whereas epigenetic changes at 11p15 are the most common aberration. We analyzed long interspersed element-1 (LINE-1) methylation levels in the blastemal component of WT and normal kidney samples to explore their prognostic significance.ResultsWT samples presented a hypomethylated pattern at all five CpG sites compared to matched normal kidney samples; therefore, the averaged methylation levels of the five CpG sites were used for further analyses. WT presented a hypomethylation profile (median 65.0%, 47.4–73.2%) compared to normal kidney samples (median 71.8%, 51.5–77.5%; p < 0.0001). No significant associations were found between LINE-1 methylation levels and clinical–pathological characteristics. We observed that LINE-1 methylation levels were lower in tumor samples from patients with relapse (median methylation 60.5%) compared to patients without relapse (median methylation 66.5%; p = 0.0005), and a receiving operating characteristic curve analysis was applied to verify the ability of LINE-1 methylation levels to discriminate WT samples from these patients. Using a cut-off value of 62.71% for LINE-1 methylation levels, the area under the curve was 0.808, with a sensitivity of 76.5% and a specificity of 83.3%. Having identified differences in LINE-1 methylation between WT samples from patients with and without relapse in this cohort, we evaluated other prognostic factors using a logistic regression model. This analysis showed that in risk stratification, LINE-1 methylation level was an independent variable for relapse risk: the lower the methylation levels, the higher the risk of relapse. The logistic regression model indicated a relapse risk increase of 30% per decreased unit of methylation (odds ratio 1.30; 95% confidence interval 1.07–1.57).ConclusionOur results reinforce previous data showing a global hypomethylation profile in WT. LINE-1 methylation levels can be suggested as a marker of relapse after chemotherapy treatment in addition to risk classification, helping to guide new treatment approaches.Electronic supplementary materialThe online version of this article (10.1186/s13148-017-0431-6) contains supplementary material, which is available to authorized users.
INTRODUCTIONChildhood renal tumors account for ~7% of all childhood cancers, and most cases are Wilms tumors (WT) or nephroblastomas (~90%), affecting one in 10,000 children under the age of fifteen 1 . It is estimated there are 500 new cases of WT every year in Brasil, with a median age-adjusted incidence rate of 9.5 cases per million 2 . The peak incidence is between the ages of 2 and 3 years 1 , but bilateral cases and those associated with congenital syndromes (5 to 10% of the cases) are diagnosed earlier 3 .Non-WT renal tumors include clear cell sarcoma of the kidney (CCSK, 2-3%, 1% of the cases), with SUMMARY Childhood renal tumors account for ~7% of all childhood cancers, and most cases are embryonic Wilms' tumors (WT). Children with WT are usually treated by either COG or SIOP. The later treats the children using preoperative chemotherapy, but both have around 90% of overall survival in five years. WT is a genetically heterogeneous group with a low prevalence of known somatic alterations. Only around 30% of the cases present mutation in known genes, and there is a relatively high degree of intra-tumor genetic heterogeneity (ITGH). Besides potentially having an impact on the clinical outcome of patients, ITGH may interfere with the search for molecular markers that are prospectively being tested by COG and SIOP. In this review, we present the proposal of the current UMBRELLA SIOP Study 2017/Brazilian Renal Tumor Group that requires the multi-sampling collection of each tumor to better evaluate possible molecular markers, as well as to understand WT biology KEYWORDS: Wilms tumor. Biomarkers. Genetic Heterogeneity PEREIRA, B, M. S. ET AL 1497 REV ASSOC MED BRAS 2019; 65(12):1496-1501in TP53 was suggested to be an independent poor prognostic factor 16 . MYCN gain was associated with anaplasia and with poorer relapse-free and overall survival, independently of tumor histology 17 . A gain of 1q is found in up to 30% of WT cases and was considered a potential prognostic biomarker regardless of the treatment protocol (COG or SIOP). A gain of 1q was associated with poorer event-free and overall survival, and, if validated, it could be used to select patients who were first treated with surgery or chemotherapy for more aggressive treatment 18-20 . Loss of heterozygosity (LOH) of both 1p and 16q were associated with lower event-free and overall survival in WT treated with surgery first 21,22 . Following these findings, for the first time, the presence of molecular alterations was used to direct therapy in WT. COG intensified the treatment for stage III/IV WT, with loss of heterozygosity (LOH) of 16q, and 1p significant improving the event-free survival 23 .All potential biomarkers discussed here were studied in a single sample from each case without considering the existence of ITGH in WT.
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