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MGMT promoter methylation testing is required for prognosis and predicting temozolomide response in gliomas. Accurate results depend on sufficient tumor cellularity, but histologic estimates of cellularity are subjective. We sought to determine whether driver mutation variant allelic frequency (VAF) could serve as a more objective metric for cellularity and identify possible false-negative MGMT samples. Among 691 adult-type diffuse gliomas, MGMT promoter methylation was assessed by pyrosequencing (N = 445) or DNA methylation array (N = 246); VAFs of TERT and IDH driver mutations were assessed by next generation sequencing. MGMT results were analyzed in relation to VAF. By pyrosequencing, 56% of all gliomas with driver mutation VAF ≥ 0.325 had MGMT promoter methylation, versus only 37% with VAF < 0.325 (p < 0.0001). The mean MGMT promoter pyrosequencing score was 19.3% for samples with VAF VAF ≥ 0.325, versus 12.7% for samples with VAF < 0.325 (p < 0.0001). Optimal VAF cutoffs differed among glioma subtypes (IDH wildtype glioblastoma: 0.12-0.18, IDH mutant astrocytoma: ~0.33, IDH mutant and 1p/19q co-deleted oligodendroglioma: 0.3-0.4). Methylation array was more sensitive for MGMT promoter methylation at lower VAFs than pyrosequencing. Microscopic examination tended to overestimate tumor cellularity when VAF was low. Re-testing low-VAF cases with methylation array and droplet digital PCR (ddPCR) confirmed that a subset of them had originally been false-negative. We conclude that driver mutation VAF is a useful quality assurance metric when evaluating MGMT promoter methylation tests, as it can help identify possible false-negative cases., (© 2023. The Author(s).)

Chromosomal instability (CIN) is a fundamental property of cancer and a key underlying mechanism of tumorigenesis and malignant progression, and has been documented in a wide variety of cancers, including colorectal carcinoma with mutations in genes such as APC. Recent reports have demonstrated that CIN, driven in part by mutations in genes maintaining overall genomic stability, is found in subsets of adult-type diffusely infiltrating gliomas of all histologic and molecular grades, with resulting elevated overall copy number burden, chromothripsis, and poor clinical outcome. Still, relatively few studies have examined the effect of this process, due in part to the difficulty of routinely measuring CIN clinically. Herein, we review the underlying mechanisms of CIN, the relationship between chromosomal instability and malignancy, the prognostic significance and treatment potential in various cancers, systemic disease, and more specifically, in diffusely infiltrating glioma subtypes. While still in the early stages of discovery compared to other solid tumor types in which CIN is a known driver of malignancy, the presence of CIN as an early factor in gliomas may in part explain the ability of these tumors to develop resistance to standard therapy, while also providing a potential molecular target for future therapies., (© 2022. The Author(s).)

DNA methylation of cytosines in CpG sites throughout the genome is an epigenetic mark contributing to gene expression regulation. DNA methylation patterns are specific to tissue type, conserved throughout life and reflect changes during tumorigenesis. DNA methylation recently emerged as a diagnostic tool to classify tumors based on a combination of preserved developmental and mutation induced signatures. In addition to the tumor classification, DNA methylation data can also be used to evaluate copy number variation, assess promoter methylation status of specific genes, such as MGMT or MLH1, and deconvolute the tumor microenvironment, assessing the tumor immune infiltrate as a potential biomarker for immunotherapy. Here we review the role for DNA methylation in tumor diagnosis., (© 2022. The Author(s).)

We present the case of a 41-year-old man who developed worsening mid-thoracic back pain and imaging revealed a well-circumscribed intramedullary tumor in the thoracic spinal cord. Subtotal resection was performed, and histopathological analysis showed a cytologically bland, minimally proliferative glial neoplasm. Sequencing revealed H3 K27M and an activating PTPN11 mutation. Serial imaging revealed slow tumor regrowth over a three year period which prompted a second resection. The recurrent tumor displayed a similar low grade-appearing histology and harbored the same H3 K27M and PTPN11 mutations as the primary. While the prognostic importance of isolated H3 K27M in spinal gliomas is well-known, the combination of these two mutations in spinal low grade glioma has not been previously reported. Importantly, PTPN11 is a component of the MAPK signaling pathway. Thus, as building evidence shows that low grade-appearing gliomas harboring H3 K27M mutations along with BRAF or FGFR1 mutations have a relatively more favorable course compared to isolated H3 K27M-mutant midline gliomas, the present case provides new evidence for the prognostic importance of activating mutations in other components of the MAPK signaling pathway. This case further highlights the importance of clinico-radio-pathologic correlation when incorporating evolving genetic data into the integrated diagnosis of rare neuroepithelial tumors., (© 2022. The Author(s).)

Chordoid meningioma is a morphological variant of meningioma designated as WHO grade 2. However, the recurrence rates varied widely in different case series, and to date, a unifying molecular genetic signature has not been identified. Among 1897 meningiomas resected at our institution, we identified 12 primary chordoid meningiomas from 12 patients. Histologically, all 12 cases had predominant (> 50%) chordoid morphology. Ten were otherwise grade 1, and two were also atypical. We performed DNA global methylation profile, copy number variation analysis, and targeted next-generation sequencing on 11 chordoid meningiomas, and compared to those of 51 non-chordoid, mostly high grade meningiomas. The chordoid meningiomas demonstrated a unique methylation profile in tSNE, UMAP, and hierarchical heatmap clustering analyses of the most differentially methylated CpGs. The most common copy number variation in chordoid meningioma was loss of 1p (7/11, 64%). Three chordoid meningiomas had 2p loss, which was significantly higher than the non-chordoid control cohort (27% vs 7.2%, p = 0.035). 22q loss was only seen in the two cases with additional atypical histological features. Chordoid meningiomas were enriched in mutations in chromatin remodeling genes EP400 (8/11,73%) KMT2C (4/11, 36%) and KMT2D (4/11, 36%), and showed low or absent NF2, TERT, SMO, and AKT1 mutations. Prognosis wise, only one case recurred. This case had atypical histology and high-grade molecular features including truncating NF2 mutation, 1p, 8p, 10, 14, 22q loss, and homozygous deletion of CDKN2A/B. Progression free survival of chordoid, otherwise grade 1 meningioma was comparable to non-chordoid WHO grade 1 meningioma (p = 0.75), and significantly better than chordoid WHO grade 2 meningioma (p = 0.019). Conclusion: the chordoid histology alone may not justify a universal WHO grade 2 designation. Screening for additional atypical histological or molecular genetic features is recommended., (© 2022. The Author(s).)

Glioblastoma (GBM) is the most common malignant primary central nervous system (CNS) neoplasm in adults, and has an almost universally poor prognosis. Recently, an emphasis on genetic and epigenetic profiling has revealed a number of molecular features useful in the diagnostic and prognostic classification of GBM, advancing our understanding of the underlying features that make these tumors so aggressive and providing the rationale for the creation of better targeted therapeutics. One such method, DNA methylation profiling, has recently emerged as an important technique for the classification of CNS tumors, with diagnostic accuracy in some cases surpassing traditional methods. However, how DNA methylation profiles change with the course of the disease remains less understood. Here, we present a case of a 30-year-old male with primary IDH-mutant GBM with widespread recurrence and death two years later. Using unsupervised hierarchical clustering of methylation probes, we created a phylogenetic map to trace the tumor path as it spread from the initial biopsy site throughout the right hemisphere, across the corpus callosum to the contralateral hemisphere, and into the brainstem. We identified molecular divergence between the right and left hemisphere GBM samples marked by distinct copy number profile alterations, alterations in specific methylation sites, and regional loss of MGMT promoter methylation, providing a potential mechanism for treatment resistance in this case. In summary, this case both highlights the molecular diversity in GBM, and illustrates a novel use for methylation profiling in establishing a phylogenetic profile to allow for spatial mapping of tumor progression.

Retinoblastoma is a childhood cancer of the retina involving germline or somatic alterations of the RB Transcriptional Corepressor 1 gene, RB1. Rare cases of sellar-suprasellar region retinoblastoma without evidence of ocular or pineal tumors have been described. A nine-month-old male presented with a sellar-suprasellar region mass. Histopathology showed an embryonal tumor with focal Flexner-Wintersteiner-like rosettes and loss of retinoblastoma protein (RB1) expression by immunohistochemistry. DNA array-based methylation profiling confidently classified the tumor as pineoblastoma group A/intracranial retinoblastoma. The patient was subsequently enrolled on an institutional translational cancer research protocol and underwent comprehensive molecular profiling, including paired tumor/normal exome and genome sequencing and RNA-sequencing of the tumor. Additionally, Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) sequencing was performed from comparator normal and disease-involved tissue to resolve complex structural variations. RNA-sequencing revealed multiple fusions clustered within 13q14.1-q21.3, including a novel in-frame fusion of RB1-SIAH3 predicted to prematurely truncate the RB1 protein. SMRT sequencing revealed a complex structural rearrangement spanning 13q14.11-q31.3, including two somatic structural variants within intron 17 of RB1. These events corresponded to the RB1-SIAH3 fusion and a novel RB1 rearrangement expected to correlate with the complete absence of RB1 protein expression. Comprehensive molecular analysis, including DNA array-based methylation profiling and sequencing-based methodologies, were critical for classification and understanding the complex mechanism of RB1 inactivation in this diagnostically challenging tumor.

Fusions involving neurotrophic tyrosine receptor kinase (NTRK) genes are detected in ≤2% of gliomas and can promote gliomagenesis. The remarkable therapeutic efficacy of TRK inhibitors, which are among the first Food and Drug Administration-approved targeted therapies for NTRK-fused gliomas, has generated significant clinical interest in characterizing these tumors. In this multi-institutional retrospective study of 42 gliomas with NTRK fusions, next generation DNA sequencing (n = 41), next generation RNA sequencing (n = 1), RNA-sequencing fusion panel (n = 16), methylation profile analysis (n = 18), and histologic evaluation (n = 42) were performed. All infantile NTRK-fused gliomas (n = 7) had high-grade histology and, with one exception, no other significant genetic alterations. Pediatric NTRK-fused gliomas (n = 13) typically involved NTRK2, ranged from low- to high-histologic grade, and demonstrated histologic overlap with desmoplastic infantile ganglioglioma, pilocytic astrocytoma, ganglioglioma, and glioblastoma, among other entities, but they rarely matched with high confidence to known methylation class families or with each other; alterations involving ATRX, PTEN, and CDKN2A/2B were present in a subset of cases. Adult NTRK-fused gliomas (n = 22) typically involved NTRK1 and had predominantly high-grade histology; genetic alterations involving IDH1, ATRX, TP53, PTEN, TERT promoter, RB1, CDKN2A/2B, NF1, and polysomy 7 were common. Unsupervised principal component analysis of methylation profiles demonstrated no obvious grouping by histologic grade, NTRK gene involved, or age group. KEGG pathway analysis detected methylation differences in genes involved in PI3K/AKT, MAPK, and other pathways. In summary, the study highlights the clinical, histologic, and molecular heterogeneity of NTRK-fused gliomas, particularly when stratified by age group.

Since the discovery that IDH1/2 mutations confer a significantly better prognosis in astrocytomas, much work has been done to identify other molecular signatures to help further stratify lower-grade astrocytomas and glioblastomas, with the goal of accurately predicting clinical outcome and identifying potentially targetable mutations. In the present study, we subclassify 135 astrocytomas (67 IDH-wildtype and 68 IDH-mutant) from The Cancer Genome Atlas dataset (TCGA) on the basis of grade, IDH-status, and the previously established prognostic factors, CDK4 amplification and CDKN2A/B deletion, within the IDH-mutant groups. We analyzed these groups for total copy number variation (CNV), total mutation burden, chromothripsis, specific mutations, and amplifications/deletions of specific genes/chromosomal regions. Herein, we demonstrate that across all of these tumor groups, total CNV level is a relatively consistent prognostic factor. We also identified a trend towards increased levels of chromothripsis in tumors with lower progression-free survival (PFS) and overall survival (OS) intervals. While no significant differences were identified in overall mutation load, we did identify a significantly higher number of cases with mutations in genes with functions related to maintaining genomic stability in groups with higher mean CNV and worse PFS and OS intervals, particularly in the IDH-mutant groups. Our data further support the case for total CNV level as a potential prognostic factor in astrocytomas, and suggest mutations in genes responsible for overall genomic instability as a possible underlying mechanism for some astrocytomas with poor clinical outcome.

Background: Extraneural metastases are relatively rare manifestations of medulloblastoma., Case Presentation: We present the case of a young boy with group three MYCN-amplified medulloblastoma. He received multimodal chemotherapy consisting of gross total resection followed by postoperative craniospinal radiation and adjuvant chemotherapy. The patient developed extraneural metastases 4 months after the end of therapy. Literature review identifies the poor prognosis of MYCN-amplified medulloblastomas as well as extraneural metastases; we review the current limitations and future directions of medulloblastoma treatment options., Conclusion: To the best of our knowledge, this is the first molecularly characterized report of extraneural metastases of medulloblastoma in a child.