Harmonization of postmortem donations for pediatric brain tumors and molecular characterization of diffuse midline gliomas

Abstract

Children diagnosed with brain tumors have the lowest overall survival of all pediatric cancers. Recent molecular studies have resulted in the discovery of recurrent driver mutations in many pediatric brain tumors. However, despite these molecular advances, the clinical outcomes of high grade tumors, including H3K27M diffuse midline glioma (H3K27M DMG), remain poor. To address the paucity of tissue for biological studies, we have established a comprehensive protocol for the coordination and processing of donated specimens at postmortem. Since 2010, 60 postmortem pediatric brain tumor donations from 26 institutions were coordinated and collected. Patient derived xenograft models and cell cultures were successfully created (76% and 44% of attempts respectively), irrespective of postmortem processing time. Histological analysis of mid-sagittal whole brain sections revealed evidence of treatment response, immune cell infiltration and the migratory path of infiltrating H3K27M DMG cells into other midline structures and cerebral lobes. Sequencing of primary and disseminated tumors confirmed the presence of oncogenic driver mutations and their obligate partners. Our findings highlight the importance of postmortem tissue donations as an invaluable resource to accelerate research, potentially leading to improved outcomes for children with aggressive brain tumors.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Coordination characteristics of postmortem donation program at CNH. (a) Flow chart of personnel and steps involved for coordination. (b) Patient demographics, types of brain tumors collected, logistics, and generation of postmortem preclinical models. Flow chart graphics were made using Adobe Illustrator.

Figure 2

Specimens collected, comprehensive whole brain processing, and utilization of specimens for preclinical models. (a) Detailed figure of processing in a patient with DMG using different platforms and utilization of tumor and healthy regions for frozen, FFPE, preclinical model generation (cell culture or xenograft) and DMSO preservation of whole brain. (b) RNA integrity was not affected by postmortem tissue collection time in DMG tumors. (c) Tissue collected for DMSO preservation representing multiple neuroanatomical locations for future generation of preclinical models. Graphics were made using Adobe Illustrator.

Figure 3

Characterization and fidelity of DMG preclinical models derived from postmortem tissue. (a) Schematic for generation of preclinical models (xenografts or primary cells) from fresh or DMSO cryopreserved tissue. (b) Oncoplot showing preclinical models generated and genomic aberrations of primary tumor obtained from WGS. (c) Characteristics of primary tumor and a matched primary cell line and xenograft model derived from postmortem tissue harboring H3.3K27M. Scale Bar = 100 µm. Schematic graphics were created using Adobe Illustrator.

Figure 4

Identification of patterns of tumor dissemination in H3K27M DMG. (a) Mid-sagittal histological characterization for identification of tumor dissemination (shown by asterisk*) as assessed by a neuropathologist. (b) H&E images of specific locations confirming presence of tumor. (c) Mutational profile identified in primary tumor and other neuroanatomical locations show affected oncogenic pathways (not all locations mentioned in the oncoplot have tumors as per histology or mutational profile). Scale Bar = 100 µm Final figure was combined on Adobe Illustrator.