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Ependymoma (EPN) is the third most common brain tumor in children and frequently recurs. Here, we report an integrated longitudinal analysis of epigenetic, genetic and tumorigenic changes in 30 patient-matched repeated relapses obtained from 10 pediatric patients to understand the mechanism of recurrences. Genome-wide DNA methylation analysis revealed stable molecular subtypes and convergent epigenetic reprogramming during serial relapses of the 5 RELA and 5 PFA EPNs that paralleled with elevated patient-derived orthotopic xenograft (PDOX) (13/27) formation in the late relapses. Differentially methylated CpGs (DMCs) preexisted in the primary tumors and persisted in the relapses (driver DMCs) were detected, ranging from 51 hypo-methylated in RELA to 148 hyper-methylated DMCs in PFA tumors; while newly acquired DMCs sustained in all the relapses but was absent in the primary tumors (booster DMCs) ranged from 38- 323 hyper-methylated DMCs in RELA and PFA EPNs, respectively. Integrated analysis of these DMC associated DNA methylation regions (DMRs) and RNAseq in both patient and PDOX tumors identified a small fraction of the differentially expressed genes (4.6±4.4% in RELA and 4.5±1.1% in PFA) as regulated by driver DMRs (e.g., up-regulated CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and booster DMRs (including the sole upregulated PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). Most these genes were novel to EPN relapses. Seven DMCs in RELA and 22 in PFA tumors were also identified as potential relapse predictors. Finally, integrating DNA methylation with histone modification identified LSD1 as a relapse driver gene. Combined treatment of a novel inhibitor SYC-836 with radiation significantly prolonged survival times in two PDOX models of recurrent PFA. This high-resolution epigenetic and genetic roadmap of EPN relapse and our 13 new PDOX models should significantly facilitate biological and preclinical studies of pediatric EPN recurrences.

Immunodeficient patients with severe burn injuries are extremely susceptible to infection with Candida albicans. In addition to Th1 cells, IL-17-producing CD4(+) T cells (Th17 cells) have recently been described as an important effector cell in host anti-Candida resistance. In this study, therefore, we tried to induce Th17 cells in cultures of severely burned patient PBMC by stimulation with the C. albicans Ag (CAg). In the results, the biomarkers for Th17 cells (IL-17 production and intracellular expression of IL-17 and retinoic acid receptor-related orphan receptor γt) were not displayed by burn patient PBMC stimulated with CAg, whereas these biomarkers of Th17 cells were detected in cultures of healthy donor PBMC stimulated with CAg. Burn patient sera were shown to be inhibitory on CAg-stimulated Th17 cell generation in healthy donor PBMC cultures; however, Th17 cells were induced by CAg in healthy donor PBMC cultures supplemented with burn patient sera that were previously treated with anti-IL-10 mAb. Also, the biomarkers of Th17 cells were not induced by CAg in healthy donor PBMC cultures supplemented with rIL-10. IL-10 was detected in serum specimens derived from severely burned patients. These results indicate that Th17 cells are not generated in burn patient PBMC cultures supplemented with CAg. IL-10, produced in response to burn injuries, is shown to be inhibitory on Th17 cell generation. The high susceptibility of severely burned patients to C. albicans infection might be influenced if burn-associated IL-10 production is intervened.

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Brain tumors are the leading cause of cancer related death in children, and there is biological differences between treatment naïve tumors and their therapy resistant relapses. As surgery is rarely an option for terminal brain tumors that have failed existing therapies, autopsy is frequently the only chance to obtain tumor tissues. Despite the urgent need of models of terminal brain tumors, it remains unknown if autopsied tumors can be used for in vitro and in vivo model development. To determine if (some) tumor cells can survival postmortem anoxia/starvation to proliferate in vitro and, more importantly, to form orthotopic xenograft tumors, we collected 29 autopsied brain tumors, including 15 diffuse intrinsic pontine gliomas (DIPG), 7 glioblastomas (GBM), 4 medulloblastomas (MB), 2 ependymoma and 1 atypical teratoid/rhabdoid tumor (ATRT), and made the following discoveries. i) A small fraction of tumor cells exhibited strong survival capacity and remained viable, ranging from 0.5%-40% (13.5% ± 10.9%), in tumors harvested 5-72 hrs (31.2 ± 25 hrs) after patients’ death. Cell viability, however, did not appear to be strictly correlated with the length of autopsy time (r = 0.17), suggesting that ample time should be given to the families to say goodbye to their loved ones. ii) While many autopsied tumor cells appear to have stayed alive for short terms in culture, only one pair of permanent lines (monolayer in FBS media and neurosphere in serum-free media) were established from a GBM tumor. iii) Direct implantation of tumor cells into matched locations in the brains of SCID mice led to the formation of xenograft tumors in 17 of 22 tumors (7 additional tumors are still pending). 8 of the 17 autopsy derived orthotopic (ADOX) models have been subtransplanted in mouse brains for > 3 times (5 DIPGs and 1 each of GBM, MB and ATRT). Detailed characterization confirmed their replication of histopathological features and genetic/genomic abnormalities of the patient tumors. iv) To identify the cellular origin of surviving patient tumor cells that propagated ADOX model formation, time course analysis of putative cancer stem cells (CD133, CD15, CD24/CD44, CD57, CD117) was performed. CD57+ tumor cells were found to be the most abundant subpopulations in the autopsied tumors, and their fractions increased in the ADOX tumors during serial subtransplantations. Additionally, CD57+ cells alone were able to form orthotopic xenografts, establishing their role as cancer stem cells. In conclusion, we have demonstrated that a fraction of tumor cells can survive the lengthy period of postmortem anoxia/starvation and regain tumorigenic capabilities in mouse brains; and identified CD57 as a new marker of the therapy-resistant cells with extraordinary survival and tumorigenic (TREST) capabilities. This novel panel of ADOX models should facilitate biological studies and preclinical drug screening of therapy-resistant pediatric brain tumors. Citation Format: Lin Qi, Baxter A. Patricia, Kogiso Mari, Du Yuchen, Lindsay Holly, Liu Zhigang, Xiumei Zhao, Yujing Zhang, Jack MF Su, Adekunle Adesina, Andrew W. Walter, Jeffery Murray, Rene McNall-Knapp, Javad Nazarian, Will Parsons, Murali Chintagumpala, Susan Blaney, Xiao-Nan Li. Autopsy derived orthotopic xenograft (ADOX) mouse models for terminal pediatric brain tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2015-1450