Recurrence is frequent in pediatric ependymoma (EPN). Our longitudinal integrated analysis of 30 patient-matched repeated relapses (3.67 ± 1.76 times) over 13 years (5.8 ± 3.8) reveals stable molecular subtypes (RELA and PFA) and convergent DNA methylation reprogramming during serial relapses accompanied by increased orthotopic patient derived xenograft (PDX) (13/27) formation in the late recurrences. A set of differentially methylated CpGs (DMCs) and DNA methylation regions (DMRs) are found to persist in primary and relapse tumors (potential driver DMCs) and are acquired exclusively in the relapses (potential booster DMCs). Integrating with RNAseq reveals differentially expressed genes regulated by potential driver DMRs (CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and potential booster DMRs (PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). DMCs predicators of relapse are also identified in the primary tumors. This study provides a high-resolution epigenetic roadmap of serial EPN relapses and 13 orthotopic PDX models to facilitate biological and preclinical studies.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Check for updates nature communications (2022) 13:7871 | 1 1234567890():,; 1234567890():,;

No abstract

Pediatric ependymoma has a propensity of developing late and multiple relapses over many years. About 50% of patients will experience relapses and eventually succumb to their disease. Our study is aimed to understand the mechanism of resistance and drivers associated with pediatric ependymoma relapse. We developed 10 sets of patient-derived orthotopic xenograft (PDOX) models of recurrent pediatric ependymoma from both RELA and PFA tumors. Time from primary tumor to last recurrence ranges from 2.75 – 13 years. Number of recurrences per patient ranges from 1 – 7 times. We performed Reduced Representation Bisulfite Sequencing (RRBS) and Whole Genome Bisulfite Sequencing (WGBS) to map the DNA methylation landscape of total of 30 samples of matched primary and recurrent tumors. Molecular subtypes and DNA methylation profiles were maintained, and RELA/PFA signature genes showed similar expression pattern during serial relapses. RELA- and PFA-specific Differentially Methylated CpGs (DMCs) are identified from primary tumors. During the recurrent process, individual patients displayed consistent changes of DMCs and shared DMCs among patients became convergent. We then identified shared common specific DMCs in recurrent RELA and PFA tumors that emerged as the driver signatures. We found that these recurrent DNA methylation signatures could be identified from primary tumors. Our analysis of the PDOX models showed that they can mostly recapitulate humor tumors’ DNA methylation and we were able to identify shared recurrent specific DMCs associated genes in PDOX models. Our comprehensive data is the first of its kind aimed to investigate the epigenetic mechanisms during pediatric ependymoma recurrence.