11p15 Epimutations in Pediatric Embryonic Tumors: Insights from a Methylome Analysis

SIMPLE SUMMARY: Epigenetics encompasses changes in DNA without changing the DNA sequence itself, and it includes, among other modifications, DNA methylation. DNA methylation consists of the addition of a methyl group (-CH3), usually to a cytosine that precedes a guanine, forming a CpG site, and it is associated with the regulation of gene expression. The disruption of CpG site methylation is widely studied in cancer. This study evaluated the methylation status of a group of CpG sites located at 11p15, which is an imprinted region. There are several imprinted regions in the human genome; they are characterized by being controlled by DNA methylation, allowing only one allele to be expressed. The 11p15 region contains two imprinted control regions (ICR1 and 2) whose changes are considered the underlying mechanism behind Beckwith–Wiedmann syndrome; patients with this syndrome present an increased risk of developing embryonic tumors. Thus, the 11p15 methylation status was evaluated in a series of sporadic embryonic tumors, revealing that a loss of imprinting may happen via the gain or loss of DNA methylation at ICR1 and/or ICR2 and in specific tumor types. ABSTRACT: Embryonic tumors share few recurrent mutations, suggesting that other mechanisms, such as aberrant DNA methylation, play a prominent role in their development. The loss of imprinting (LOI) at the chromosome region 11p15 is the germline alteration behind Beckwith–Wiedemann syndrome that results in an increased risk of developing several embryonic tumors. This study analyzed the methylome, using EPIC Beadchip arrays from 99 sporadic embryonic tumors. Among these tumors, 46.5% and 14.6% presented alterations at imprinted control regions (ICRs) 1 and 2, respectively. Based on the methylation levels of ICR1 and ICR2, four clusters formed with distinct methylation patterns, mostly for medulloblastomas (ICR1 loss of methylation (LOM)), Wilms tumors, and hepatoblastomas (ICR1 gain of methylation (GOM), with or without ICR2 LOM). To validate the results, the methylation status of 29 cases was assessed with MS-MLPA, and a high level of agreement was found between both methodologies: 93% for ICR1 and 79% for ICR2. The MS-MLPA results indicate that 15 (51.7%) had ICR1 GOM and 11 (37.9%) had ICR2 LOM. To further validate our findings, the ICR1 methylation status was characterized via digital PCR (dPCR) in cell-free DNA (cfDNA) extracted from peripheral blood. At diagnosis, we detected alterations in the methylation levels of ICR1 in 62% of the cases, with an agreement of 76% between the tumor tissue (MS-MLPA) and cfDNA methods. Among the disagreements, the dPCR was able to detect ICR1 methylation level changes presented at heterogeneous levels in the tumor tissue, which were detected only in the methylome analysis. This study highlights the prevalence of 11p15 methylation status in sporadic embryonic tumors, with differences relating to methylation levels (gain or loss), location (ICR1 or ICR2), and tumor types (medulloblastomas, Wilms tumors, and hepatoblastomas).