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.
Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/pathology , Glioma/pathology , Histones/genetics , Mutation , Adolescent , Adult , Animals , Autopsy , Brain Neoplasms/genetics , Child , Child, Preschool , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioma/genetics , Humans , Infant , Male , Mice, Inbred NOD , Mice, SCID , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Young AdultABSTRACT
Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.
Subject(s)
Body Fluids/metabolism , Circulating Tumor DNA/metabolism , Biomarkers, Tumor/genetics , Humans , Limit of Detection , Mutation , Polymerase Chain ReactionABSTRACT
Diffuse intrinsic pontine gliomas (DIPGs) are deadly tumors comprising 10%-15% of all childhood CNS cancers. Standard treatment is considered palliative and prognosis is near universal mortality. DIPGs have been classified into genomic subtypes based on histone variants with the lysine to methionine mutation on position 27 of histone tails (K27M). Given the increasing promise of immunotherapy, there have been ongoing efforts to identify tumor-specific antigens to serve as immunologic targets. We evaluated a large cohort of CNS specimens for Wilms' tumor protein (WT1) expression. These specimens include primary pediatric CNS tumors (n = 38 midline gliomas and n = 3 non-midline gliomas; n = 23 DIPG, n = 10 low-grade gliomas, n = 8 high-grade gliomas), and DIPG primary cells. Here, we report the validation of WT1 as a tumor-associated antigen in DIPGs. We further report that WT1 expression is significantly correlated with specific oncohistone variants, with the highest expression detected in the H3.3K27M subgroup. WT1 expression was absent in all control specimens (n = 21). Western blot assays using DIPG primary cells (n = 6) showed a trend of higher WT1 expression in H3.3K27M cells when compared with H3.1 K27M cells and H3 wildtype cells. Our data are the first indication of the association between WT1 and DIPG, with specific upregulation in those harboring oncohistone H3.3K27M.
Subject(s)
Brain Stem Neoplasms/metabolism , Diffuse Intrinsic Pontine Glioma/metabolism , Gene Expression Regulation, Neoplastic , WT1 Proteins/biosynthesis , Adolescent , Brain Stem Neoplasms/genetics , Brain Stem Neoplasms/pathology , Child , Child, Preschool , Cohort Studies , Diffuse Intrinsic Pontine Glioma/genetics , Diffuse Intrinsic Pontine Glioma/pathology , Female , Humans , Infant , Male , Mutation/genetics , WT1 Proteins/genetics , Young AdultABSTRACT
Anaplastic astrocytomas are aggressive glial cancers that present poor prognosis and high recurrence. Heterozygous IDH1 R132H mutations are common in adolescent and young adult anaplastic astrocytomas. In a majority of cases, the IDH1 R132H mutation is unique to the tumor, although rare cases of anaplastic astrocytoma have been described in patients with mosaic IDH1 mutations (Ollier disease or Maffucci syndrome). Here, we present two siblings with IDH1 R132H mutant high grade astrocytomas diagnosed at 14 and 26 years of age. Analysis of IDHR132H mutations in the siblings' tumors and non-neoplastic tissues, including healthy regions of the brain, cheek cells, and primary teeth indicate mosaicism of IDHR132H. Whole exome sequencing of the tumor tissue did not reveal any other common mutations between the two siblings. This study demonstrates the first example of IDH1 R132H mosaicism, acquired during early development, that provides an alternative mechanism of cancer predisposition.
