RESUMEN
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).
RESUMEN
Pediatric tumors share few recurrent mutations and are instead characterized by copy number alterations (CNAs). The cell-free DNA (cfDNA) is a prominent source for the detection of cancer-specific biomarkers in plasma. We profiled CNAs in the tumor tissues for further evaluation of alterations in 1q, MYCN and 17p in the circulating tumor DNA (ctDNA) in the peripheral blood at diagnosis and follow-up using digital PCR. We report that among the different kinds of tumors (neuroblastoma, Wilms tumor, Ewing sarcoma, rhabdomyosarcoma, leiomyosarcoma, osteosarcoma and benign teratoma), neuroblastoma presented the greatest amount of cfDNA, in correlation with tumor volume. Considering all tumors, cfDNA levels correlated with tumor stage, metastasis at diagnosis and metastasis developed during therapy. In the tumor tissue, at least one CNA (at CRABP2, TP53, surrogate markers for 1q and 17p, respectively, and MYCN) was observed in 89% of patients. At diagnosis, CNAs levels were concordant between tumor and ctDNA in 56% of the cases, and for the remaining 44%, 91.4% of the CNAs were present only in cfDNA and 8.6% only in the tumor. Within the cfDNA, we observed that 46% and 23% of the patients had MYCN and 1q gain, respectively. The use of specific CNAs as targets for liquid biopsy in pediatric patients with cancer can improve diagnosis and should be considered for monitoring of the disease response.