Faculty Bibliography

Central nervous system (CNS) tumor with BCOR internal tandem duplication (BCOR-ITD) have recently been introduced in the 5th edition of the WHO classification of CNS tumors, however, their molecular makeup and clinical characteristics remain widely enigmatic. This is further complicated by the recent discovery of tumors characterized by gene fusions involving BCOR or its homologue BCORL1. We identified a cohort of 206 BCOR altered CNS tumors via DNA methylation profiling and conducted in-depth molecular and clinical characterization in an international effort. By performing t-SNE clustering analysis we found that BCOR-fusion tumors form a distinct cluster (n=61), adjacent to BCOR-ITD cases (n=145). The identified fusion partners of BCOR(L1) included EP300 (n=20), CREBBP (n=5), and NUTM2HP (n=1). Notably, three cases within the BCOR-ITD cluster harbored a c-terminal intragenic deletion within BCOR. With respect to clinical characteristics gender ratio was balanced in BCOR-fusion cases (m/f, 1.1), whereas predominance of male patients was observed in the BCOR-ITD group (m/f, 1.5). Moreover, age at diagnosis of BCOR-fusion patients was higher as compared to BCOR-ITD cases (15 vs 4.5 years). Interestingly, BCOR-fusion tumors were exclusively found in the supratentorial region being originally diagnosed as ependymomas or gliomas whereas BCOR-ITD emerged across the entire CNS with diverse original diagnoses. 8% of BCOR-ITD and none of BCOR-fusion cases were disseminated at diagnosis. In line with this observation, 40% of first relapses within the BCORITD group were metastatic which was less frequent in BCOR-fusion tumors. Survival estimates demonstrated no differences, generally showing short median PFS (BCOR-fusion, 2 years, n=15; BCOR-ITD, 1.8 years, n=55) and intermediate OS rates (BCOR-fusion, 6.8 years, n=18; BCOR-ITD 6.3 years, n=60). Further molecular and clinical characterization is ongoing potentially revealing first therapeutic leads for these highly aggressive CNS tumor types

The histone-lysine N-methyltransferase EZH2 is the catalytic component of the PRC2 complex and is overexpressed in several medulloblastoma subtypes. However, its role in medulloblastoma tumorigenesis has been shown to be context-dependent using genetic approaches. Furthermore, pharmacological approaches have been limited by the very poor blood-brain barrier (BBB) penetration of current EZH2 inhibitors in use. Using laser capture microdissection and RNA-Seq analysis of human nodular/desmoplastic SHH medulloblastoma FFPE tissue, we provide data for the spatial epigenetic heterogeneity of primitive/proliferative regions compared to nodular/mature regions. Bioinformatic analysis identifies ~120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3 or SUZ12. ChIP-Seq analysis shows striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Utilizing a genetically-engineered mouse model of SHH medulloblastoma, we show that conditional EZH2 genetic ablation within medulloblastoma cells results in wide-spread tumor cell differentiation (n=31 mice;*p=2e-07). Conversely, conditional EZH2 (Y641F) activation in this GEM model prevents tumor cell differentiation. Notably, we have found that the CDNK2A (p16) locus is an important EZH2 target that regulates tumor cell differentiation. qRT-PCR analysis of SHH medulloblastoma in wild-type and Ezh2 knockout settings show significant reduction in Gli1 and CCND1 and increase p15 and p16 expression in Ezh2 knockout mice compared to Ezh2 wildtype mice (*p<0.05). Importantly, genetic ablation of p16 conditionally in SHH MB EZH2 double knockout mice rescues the widespread tumor cell differentiation (n=9 mice;*p=3e-06) seen in Ezh2 single knockout SHH medulloblastoma mice. Finally, we developed a novel fucoidan-based nanoparticle strategy to deliver the EZH2 inhibitor (EPZ-6438) across the intact BBB of this GEM model to achieve significant extension of mouse survival (median 70 days compared to 19 days in control mice;*p=0.01, Mantel-Cox) with potential utility for other pediatric brain tumors

Pediatric high-grade gliomas (pHGGs), encompassing hemispheric and diffuse midline gliomas (DMGs), remain a devastating disease. The last decade has revealed oncogenic drivers including single nucleotide variants (SNVs) in histones. However, the contribution of structural variants (SVs) to gliomagenesis has not been systematically explored due to limitations in early SV analysis approaches. Using SV algorithms, we recently created, we analyzed SVs in whole-genome sequences of 179 pHGGs including a novel cohort of treatment naive samples-the largest WGS cohort assembled in adult or pediatric glioma. The most recurrent SVs targeted MYC isoforms and receptor tyrosine kinases, including a novel SV amplifying a MYC enhancer in the lncRNA CCDC26 in 12% of DMGs and revealing a more central role for MYC in these cancers than previously known. Applying de novo SV signature discovery, we identified five signatures including three (SVsig1-3) involving primarily simple SVs, and two (SVsig4-5) involving complex, clustered SVs. These SV signatures associated with genetic variants that differed from what was observed for SV signatures in other cancers, suggesting different links to underlying biology. Tumors with simple SV signatures were TP53 wild-type but were enriched with alterations in TP53 pathway members PPM1D and MDM4. Complex signatures were associated with direct aberrations in TP53, CDKN2A, and RB1 early in tumor evolution, and with extrachromosomal amplicons that likely occurred later. All pHGGs exhibited at least one simple SV signature but complex SV signatures were primarily restricted to subsets of H3.3K27M DMGs and hemispheric pHGGs. Importantly, DMGs with the complex SV signatures SVsig4-5 were associated with shorter overall survival independent of histone type and TP53 status. These data inform the role and impact of SVs in gliomagenesis and mechanisms that shape them

Introduction: Pineal region tumors comprise a small percentage of intracranial brain malignancies and glioblastoma (GBM) in this region is extremely rare and poorly defined. Case report: We describe a rare presentation of IDH-wildtype glioblastoma arising from the pineal region in a 49-year-old female, with unusual histological and molecular findings. The patient presented with worsening headaches and diplopia, and her MRI revealed a 3.4cm heterogeneously enhancing solid and cystic pineal gland mass, causing mass effect and hydrocephalus. Results/Discussion: The resection specimen revealed a moderately cellular and highly proliferative tumor consisting of oval-to-spindled cells with astrocytic morphology, variable amounts of eosinophilic cytoplasm and nuclear pleomorphism, embedded in a myxoid matrix with distinct chondroid appearance. Atypical microvascular proliferation and brisk mitotic activity were noted, without palisading necrosis. Immunohistochemically, tumor cells were strongly GFAP positive and negative for IDH1-R132H, P53, H3K27M, and BRAFV600E. MGMT promoter methylation was detected. Next-generation sequencing uncovered CD274 (PD-L1), PDCD1LG2 (PDL2), and JAK2 amplifications; ATM, MUTYH, CDKN2C, PIK3CA mutations; and CDKN2A/B loss. The tumor was IDH and Histone H3 wildtype, and lacked common GBM-associated alterations at loci such as TERT, PTEN, or EGFR. DNA methylation analysis best classified the tumor as glioblastoma, IDH-wildtype (0.72 score), with the subclass midline (GBM-MID, 0.68 score). GBM-MID comprises tumors with the histological diagnosis of glioblastoma and midline structures location. These tumors share epigenetic similarities with the diffuse midline glioma, H3K27M-mutant methylation cluster, but lack the characteristic histone mutation. Overall, pineal region glioblastoma is a rare disease with diagnostic and therapeutic challenges. Unlike most pineal glioblastomas reported to date, this case displayed methylated MGMT status and the patient is progression-free three months after partial resection. The unique molecular alterations discovered in this rare glioblastoma, particularly PD-L1 and PD-L2 gene amplifications, offer future therapeutic considerations in this patient for potential response to immune checkpoint inhibitors

Introduction: Ependymomas (EPN) are neuroepithelial malignancies most common in children, in which a clear association between tumor grade and patient outcome is not well established. Supratentorial ependymomas containing ZFTA gene fusions were recently established as a unique tumor entity with aggressive behavior. Fusions between ZFTA (formerly C11orf95) and its most common partner, RELA, pathologically activate the NFkappaB signaling pathway. Deletions of CDKN2A are independent predictors of poor survival in RELA-fused ZFTA fusion-positive EPN. Case report: We present the case of a 4-year-old male with an aggressive CNS WHO grade 3 ZFTA fusion-positive supratentorial ependymoma in which DNA methylation profiling was essential for rendering the correct diagnosis. The patient presented with a one-month history of emesis and was found to have a large left frontal hemorrhagic mass, causing severe hydrocephalus, midline shift, and vasogenic edema. Histopathology revealed a markedly hypercellular tumor, with pseudorosette-like perivascular arrangements, broad zones of necrosis, and focal areas of atypical microvascular hyperplasia. Tumor cells were strongly positive for GFAP and showed EMA positivity in perinuclear and ring-like patterns. Next-generation sequencing disclosed CDKN2A/B and MTAP loss; but did not detect any fusions, even though the panel tests for RELA fusions. Ultimately, DNA methylation- based profiling confidently classified the tumor as ZFTA fusion-positive EPD subgroup (calibrated score: 0.985). Over the course of one year and a short course of proton beam therapy, the patient underwent multiple resections of recurrent/residual tumor.
Discussion(s): DNA methylation-based classification or FISH break-apart probes are necessary to correctly diagnose ZFTA fusion-positive ependymomas, as commercial sequencing-based assays may fail to detect all ZFTA/ RELA fusion partners. Furthermore, since CDKN2A loss represents an independent parameter for risk stratification within ZFTA fusion-positive ependymomas, molecular analysis and/or p16 immunohistochemistry as a surrogate for homozygous CDKN2A loss, represent additional tools with diagnostic/ prognostic importance

Introduction: Gene rearrangements play a critical role in the development of brain tumors. RNA next-generation sequencing (NGS) panels cover a limited number of genes, are rarely successful in FFPE samples > 5 years old, and cannot detect rearrangements between genes and non-coding regulatory regions. We evaluated whole genome Hi-C NGS for detection of gene fusions and cryptic rearrangements.
Method(s): DNA was extracted from FFPE scrolls of 55 glial and non-glial brain tumors and processed using Arima-HiC+ FFPE Sample protocol, consisting of chromatin fragmentation, labeling, and re-ligation, followed by DNA purification and library preparation for paired-end Illumina sequencing with an average of 10X genome coverage (100M PE reads per sample). Data were analyzed using the Arima-SV pipeline using Juicer and HiCUP, SV detection using HiC-Breakfinder, loop calling using Juicer Tools, and integrative data visualization using Juicebox. Overexpression of putative driver genes was confirmed by immunohistochemistry.
Result(s): Hi-C libraries were prepared and sequenced from FFPE tissues including samples that failed RNA NGS. Hi-C successfully detected gene-gene fusions including actionable EML4-NTRK3, ETV6-NTRK3, fusions. We detected rearrangements missed by RNA NGS (i.e., complex MYBL1 rearrangement) or between non-coding regions and known cancer genes (i.e. PDL1, PAX5, NRAS, TERT, KAT6A, GATA6, and ARID1B). Since Hi-C data captures 3D genome structural features such as chromatin loops and topological domains, datasets were of high quality and capable of detecting up to 13,000 chromatin loops per tumor.
Conclusion(s): Genome-wide Hi-C NGS is successful in detecting gene fusions and cryptic rearrangements between coding and non-coding regions in archival FFPE tissue including degraded samples. Because Hi-C data captures 3D genome structures, these datasets simultaneously inform gene regulatory mechanisms that may play a role in oncogenesis or tumor progression. Whole-genome Hi-C NGS expands our ability to detect actionable and novel drivers, and potentially new therapeutic targets in a single NGS workflow

DNA methylation array profiling is an emerging modality in molecular diagnostics, which is particularly useful for cases with unclear or conflicting histology. Lesions with mixed subependymoma and ependymoma components have been well documented in the literature but grading and management of these lesions is unclear as they may represent unique entities with an intermediate prognosis to pure subependymoma and ependymomas or a histologic variant within one of these established diagnostic entities. We identified 10 cases between the years 2015-2021 that were classified as subependymoma by DNA methylation array profiling despite the given histologic diagnoses of low-grade ependymal neoplasm (1), ependymoma (5), mixed ependymomasubependymoma (2), or pilocytic astrocytoma (2). Eight of the surgical specimens were from men while 2 were from women, with the range of age at time of surgery being 30 - 75 years old (median = 57.5 years old). The majority of these tumors arose in the posterior fossa (8), including all of those with an original histologic diagnosis of ependymoma and mixed ependymoma-subependymoma, while the remaining 2 cases (1 pilocytic astrocytoma and 1 low grade ependymal neoplasm) were supratentorial. Laterality of the lesions was distributed between left (2), right (3), and midline (4). All but 2 cases were from initial resections, with one of the 2 recurrent cases having a PFS of 6 years before re-resection (unknown for the second recurrent case). Short-term follow-up data was available for 3 patients ranging from 13 - 21 months during which time none had any evidence of progression. These cases illustrate the value of methylation array profiling in characterizing ependymomas and related tumors, particularly in the posterior fossa

Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients' clinical course are unknown. We assembled a cohort of 185 tumors classified as MPE based on DNA methylation from pediatric, adolescent, and adult patients. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPEB, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. Tumors in the methylation class MPE were histologically diagnosed as WHO grade I (59%), WHO grade II (37%), or WHO grade III tumors (4%). 75/77 analyzed tumors expressed HOXB13, which is a diagnostic feature not detected in other spinal ependymal tumors. Based on DNA methylation, our series split into two subtypes. MPE-A occurred in younger patients (median age 27 vs. 45 years, p=7.3e-05). They were enriched with WHO grade I tumors and associated with papillary morphology and MGMT promoter hypermethylation (all p<0.001). MPE-B included most tumors initially diagnosed as WHO grade II and cases with tanycytic morphology. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. 15/30 MPE-A could not be totally resected compared to 1/58 MPE-B (p=6.3e-08), and progressionfree survival was significantly better for MPE-B (p=3.4e-06, 10-year relapse rate 33% vs. 85%). We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment

PURPOSE/OBJECTIVE:Lung adenocarcinoma (LUAD) is a clinically heterogenous disease, which is highlighted by the unpredictable recurrence in low-stage tumors and highly variable responses observed in patients treated with immunotherapies, which cannot be explained by mutational profiles. DNA methylation-based classification and understanding of microenviromental heterogeneity may allow stratification into clinically relevant molecular subtypes of LUADs. EXPERIMENTAL DESIGN/METHODS:We characterize the genome-wide DNA methylation landscape of 88 resected LUAD tumors. Exome sequencing focusing on a panel of cancer-related genes was used to genotype these adenocarcinoma samples. Bioinformatic and statistical tools, the immune cell composition, DNA methylation age (DNAm age), and DNA methylation clustering were used to identify clinically relevant subgroups. RESULTS:Deconvolution of DNA methylation data identified immunologically hot and cold subsets of lung adenocarcinomas. Additionally, concurrent factors were analyzed that could affect the immune microenvironment, such as smoking history, ethnicity, or presence of KRAS or TP53 mutations. When the DNAm age was calculated, a lower DNAm age was correlated with the presence of a set of oncogenic drivers, poor overall survival, and specific immune cell populations. Unsupervised DNA methylation clustering identified 6 molecular subgroups of LUAD tumors with distinct clinical and microenvironmental characteristics. CONCLUSIONS:Our results demonstrate that DNA methylation signatures can stratify lung adenocarcinoma into clinically relevant subtypes, and thus such classification of LUAD at the time of resection may lead to better methods in predicting tumor recurrence and therapy responses.