MYB/MYBL1-altered gliomas frequently harbor truncations and non-productive fusions in the MYB and MYBL1 genes.

Astrocytomas that harbor recurrent genomic alterations in MYB or MYBL1 are a group of Pediatric-type diffuse low-grade gliomas that were newly recognized in the 2021 WHO Classification of Tumors of the Central Nervous System. These tumors are described in the WHO classification as harboring fusions in MYB or MYBL1. In this report, we examine 14 consecutive cases in which a MYB or MYBL1 alteration was identified, each with diagnostic confirmation by genome-wide DNA methylation profiling (6 Angiocentric gliomas and 8 Diffuse astrocytomas, MYB- or MYBL1-altered), for their specific genomic alterations in these genes. Using RNA sequencing, we find productive in-frame fusions of the MYB or MYBL1 genes in only 5/14 cases. The remaining 9 cases show genomic alterations that result in truncation of the gene, without evidence of an in-frame fusion partner. Gene expression analysis showed overexpression of the MYB(L1) genes, regardless of the presence of a productive fusion. In addition, QKI, a recognized fusion partner common in angiocentric glioma, was generally up-regulated in these 14 cases, compared to a cohort comprising >1000 CNS tumors of various types, regardless of whether a genomic alteration in QKI was present. Overall, the results show that truncations, in the absence of a productive fusion, of the MYB(L1) genes can likely drive the tumors and have implications for the analysis and diagnosis of Angiocentric glioma and Diffuse astrocytoma, MYB- or MYBL1-altered, especially for cases that are tested on panels designed to focus on fusion detection.

A patient-derived cell model for malignant transformation in IDH-mutant glioma.

Malignant transformation (MT) is commonly seen in IDH-mutant gliomas. There has been a growing research interest in revealing its underlying mechanisms and intervening prior to MT at the early stages of the transforming process. Here we established a unique pair of matched 3D cell models: 403L, derived from a low-grade glioma (LGG), and 403H, derived from a high-grade glioma (HGG), by utilizing IDH-mutant astrocytoma samples from the same patient when the tumor was diagnosed as WHO grade 2 (tumor mutational burden (TMB) of 3.96/Mb) and later as grade 4 (TMB of 70.07/Mb), respectively. Both cell models were authenticated to a patient's sample retaining endogenous expression of IDH1 R132H. DNA methylation profiles of the parental tumors referred to LGG and HGG IDH-mutant glioma clusters. The immunopositivity of SOX2, NESTIN, GFAP, OLIG2, and beta 3-Tubulin suggested the multilineage potential of both models. 403H was more prompt to cell invasion and developed infiltrative HGG in vivo. The differentially expressed genes (DEGs) from the RNA sequencing analysis revealed the tumor invasion and aggressiveness related genes exclusively upregulated in the 403H model. Pathway analysis showcased an enrichment of genes associated with epithelial-mesenchymal transition (EMT) and Notch signaling pathways in 403H and 403L, respectively. Mass spectrometry-based targeted metabolomics and hyperpolarized (HP) 1-13C pyruvate in-cell NMR analyses demonstrated significant alterations in the TCA cycle and fatty acid metabolism. Citrate, glutamine, and 2-HG levels were significantly higher in 403H. To our knowledge, this is the first report describing the development of a matched pair of 3D patient-derived cell models representative of MT and temozolomide (TMZ)-induced hypermutator phenotype (HMP) in IDH-mutant glioma, providing insights into genetic and metabolic changes during MT/HMP. This novel in vitro model allows further investigation of the mechanisms of MT at the cellular level.

Atypical Choroid Plexus Tumor of the Cauda Equina With Metastases to the Spinal Cord and Brain.

We report a case of a 57-year-old man with a tumor arising from the cauda equina with spinal cord and intracranial metastases in the basal cisterns and along the cranial nerves. He presented with severe lower back pain and mild gait imbalance. His imaging revealed a large mass in the lumbosacral region with involvement of the cauda equina, intradural extramedullary enhancing metastases in the thoracic spinal canal, and intracranial metastases in the suprasellar cistern and along both trigeminal and facial/vestibulocochlear nerve complexes. Pathological examination of the resected thoracic spinal cord mass showed an atypical papillary proliferation with moderate nuclear pleomorphism and rare mitotic figures. While the morphologic and immunophenotypic features were consistent with the diagnosis of a choroid plexus tumor, the atypical location for this entity required the exclusion of other epithelioid tumors with papillary architecture. Additional immunohistochemical markers were used to exclude a metastatic adenocarcinoma, a papillary variant of a meningioma, and a papillary variant of an ependymoma. Ultimately, methylation-based tumor profiling determined that the methylation class was a match for "plexus tumor" resulting in the integrated diagnosis of the tumor with features of choroid plexus papilloma. This is a unique presentation for both the location and the metastatic spread. The methylation profile was instrumental in establishing this diagnosis.

Papillary tumor of the pineal region: analysis of DNA methylation profiles and clinical outcomes in 76 cases.

Papillary tumor of the pineal region (PTPR) is an uncommon tumor of the pineal region with distinctive histopathologic and molecular characteristics. Experience is limited with respect to its molecular heterogeneity and clinical characteristics. Here, we describe 39 new cases and combine these with 37 previously published cases for a cohort of 76 PTPR's, all confirmed by methylation profiling. As previously reported, two main methylation groups were identified (PTPR-A and PTPR-B). In our analysis we extended the subtyping into three subtypes: PTPR-A, PTPR-B1 and PTPR-B2 supported by DNA methylation profile and genomic copy number variations. Frequent loss of chromosome 3 or 14 was found in PTPR-B1 tumors but not in PTPR-B2. Examination of clinical outcome showed that nearly half (14/30, 47%) of examined patients experienced tumor progression with significant difference among the subtypes (p value = 0.046). Our analysis extends the understanding of this uncommon but distinct neuroepithelial tumor by describing its molecular heterogeneity and clinical outcomes, including its tendency towards tumor recurrence.

The Role of Interferon-γ in Autoimmune Polyendocrine Syndrome Type 1.

BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a life-threatening, autosomal recessive syndrome caused by autoimmune regulator (AIRE) deficiency. In APS-1, self-reactive T cells escape thymic negative selection, infiltrate organs, and drive autoimmune injury. The effector mechanisms governing T-cell-mediated damage in APS-1 remain poorly understood. METHODS: We examined whether APS-1 could be classified as a disease mediated by interferon-γ. We first assessed patients with APS-1 who were participating in a prospective natural history study and evaluated mRNA and protein expression in blood and tissues. We then examined the pathogenic role of interferon-γ using Aire-/-Ifng-/- mice and Aire-/- mice treated with the Janus kinase (JAK) inhibitor ruxolitinib. On the basis of our findings, we used ruxolitinib to treat five patients with APS-1 and assessed clinical, immunologic, histologic, transcriptional, and autoantibody responses. RESULTS: Patients with APS-1 had enhanced interferon-γ responses in blood and in all examined autoimmunity-affected tissues. Aire-/- mice had selectively increased interferon-γ production by T cells and enhanced interferon-γ, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and CXCL9 signals in multiple organs. Ifng ablation or ruxolitinib-induced JAK-STAT blockade in Aire-/- mice normalized interferon-γ responses and averted T-cell infiltration and damage in organs. Ruxolitinib treatment of five patients with APS-1 led to decreased levels of T-cell-derived interferon-γ, normalized interferon-γ and CXCL9 levels, and remission of alopecia, oral candidiasis, nail dystrophy, gastritis, enteritis, arthritis, Sjögren's-like syndrome, urticaria, and thyroiditis. No serious adverse effects from ruxolitinib were identified in these patients. CONCLUSIONS: Our findings indicate that APS-1, which is caused by AIRE deficiency, is characterized by excessive, multiorgan interferon-γ-mediated responses. JAK inhibition with ruxolitinib in five patients showed promising results. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Spatial Transcriptomic Analysis of Pituitary Corticotroph Tumors.

Context: Spatial transcriptomic (ST) analysis of tumors provides a novel approach to studying gene expression along with the localization of tumor cells in their environment to uncover spatial interactions. Design: We present ST analysis of corticotroph pituitary neuroendocrine tumors (PitNETs) from formalin-fixed, paraffin-embedded tissues. ST data were compared to immunohistochemistry results. Gene expression profiles were reviewed for cluster annotations, and differentially expressed genes were used for pathway analysis. Results: Seven tumors were used for ST analysis. In situ annotation of tumor tissue was inferred from the gene expression profiles and was in concordance with the annotation made by a pathologist. Furthermore, relative gene expression in the tumor corresponded to common protein staining used in the evaluation of PitNETs, such as reticulin and Ki-67 index. Finally, we identified intratumor heterogeneity; clusters within the same tumor may present with different transcriptomic profiles, unveiling potential intratumor cell variability. Conclusion: Together, our results provide the first attempt to clarify the spatial cell profile in PitNETs.

Virtual multi-institutional tumor board: a strategy for personalized diagnoses and management of rare CNS tumors.

PURPOSE: Multidisciplinary tumor boards (MTBs) integrate clinical, molecular, and radiological information and facilitate coordination of neuro-oncology care. During the COVID-19 pandemic, our MTB transitioned to a virtual and multi-institutional format. We hypothesized that this expansion would allow expert review of challenging neuro-oncology cases and contribute to the care of patients with limited access to specialized centers. METHODS: We retrospectively reviewed records from virtual MTBs held between 04/2020-03/2021. Data collected included measures of potential clinical impact, including referrals to observational or therapeutic studies, referrals for specialized neuropathology analysis, and whether molecular findings led to a change in diagnosis and/or guided management suggestions. RESULTS: During 25 meetings, 32 presenters discussed 44 cases. Approximately half (n = 20; 48%) involved a rare central nervous system (CNS) tumor. In 21% (n = 9) the diagnosis was changed or refined based on molecular profiling obtained at the NIH and in 36% (n = 15) molecular findings guided management. Clinical trial suggestions were offered to 31% (n = 13), enrollment in the observational NCI Natural History Study to 21% (n = 9), neuropathology review and molecular testing at the NIH to 17% (n = 7), and all received management suggestions. CONCLUSION: Virtual multi-institutional MTBs enable remote expert review of CNS tumors. We propose them as a strategy to facilitate expert opinions from specialized centers, especially for rare CNS tumors, helping mitigate geographic barriers to patient care and serving as a pre-screening tool for studies. Advanced molecular testing is key to obtaining a precise diagnosis, discovering potentially actionable targets, and guiding management.

Diffuse hemispheric glioma with H3 p.K28M (K27M) mutation: Unusual non-midline presentation of diffuse midline glioma, H3 K27M-altered?

Diffuse midline glioma, H3 K27-altered (DMG-H3 K27) is an aggressive group of diffuse gliomas that predominantly occurs in pediatric patients, involves midline structures, and displays loss of H3 p.K28me3 (K27me3) expression by immunohistochemistry and characteristic genetic/epigenetic profile. Rare examples of a diffuse glioma with an H3 p.K28M (K27M) mutation and without involvement of the midline structures, so-called "diffuse hemispheric glioma with H3 p.K28M (K27M) mutation" (DHG-H3 K27), have been reported. Herein, we describe 2 additional cases of radiologically confirmed DHG-H3 K27 and summarize previously reported cases. We performed histological, immunohistochemical, molecular, and DNA methylation analysis and provided clinical follow-up in both cases. Overall, DHG-H3 K27 is an unusual group of diffuse gliomas that shows similar clinical, histopathological, genomic, and epigenetic features to DMG-H3 K27 as well as enrichment for activating alterations in MAPK pathway genes. These findings suggest that DHG-H3 K27 is closely related to DMG-H3 K27 and may represent an unusual presentation of DMG-H3 K27 without apparent midline involvement and with frequent MAPK pathway activation. Detailed reports of additional cases with clinical follow-up will be important to expand our understanding of this unusual group of diffuse gliomas and to better define the clinical outcome and how to classify DHG-H3 K27.

Molecular and clinicopathologic characteristics of CNS embryonal tumors with BRD4::LEUTX fusion.

Central nervous system (CNS) embryonal tumors are a heterogeneous group of high-grade malignancies, and the increasing clinical use of methylation profiling and next-generation sequencing has led to the identification of molecularly distinct subtypes. One proposed tumor type, CNS tumor with BRD4::LEUTX fusion, has been described. As only a few CNS tumors with BRD4::LEUTX fusions have been described, we herein characterize a cohort of 9 such cases (4 new, 5 previously published) to further describe their clinicopathologic and molecular features. We demonstrate that CNS embryonal tumor with BRD4::LEUTX fusion comprises a well-defined methylation class/cluster. We find that patients are young (4 years or younger), with large tumors at variable locations, and frequently with evidence of leptomeningeal/cerebrospinal fluid (CSF) dissemination. Histologically, tumors were highly cellular with high-grade embryonal features. Immunohistochemically, 5/5 cases showed synaptophysin and 4/5 showed OLIG2 expression, thus overlapping with CNS neuroblastoma, FOXR2-activated. DNA copy number profiles were generally flat; however, two tumors had chromosome 1q gains. No recurring genomic changes, besides the presence of the fusion, were found. The LEUTX portion of the fusion transcript was constant in all cases assessed, while the BRD4 portion varied but included a domain with proto-oncogenic activity in all cases. Two patients with clinical follow up available had tumors with excellent response to chemotherapy. Two of our patients were alive without evidence of recurrence or progression after gross total resection and chemotherapy at 16 and 33 months. One patient relapsed, and the last of our four patients died of disease one month after diagnosis. Overall, this case series provides additional evidence for this as a distinct tumor type defined by the presence of a specific fusion as well as a distinct DNA methylation signature. Studies on larger series are required to further characterize these tumors.

Evidence of the Role of Inflammation and the Hormonal Environment in the Pathogenesis of Adrenal Myelolipomas in Congenital Adrenal Hyperplasia.

Adrenal myelolipomas (AML) are composed of mature adipose and hematopoietic components. They represent approximately 3 percent of adrenal tumors and are commonly found in patients with congenital adrenal hyperplasia (CAH). CAH provides a unique environment to explore AML pathogenesis. We aimed to evaluate the role of the immune system and hormones that accumulate in poorly controlled CAH in the development of AML. When compared to normal adrenal tissue, CAH-affected adrenal tissue and myelolipomas showed an increased expression of inflammatory cells (CD68, IL2Rbeta), stem cells (CD117) B cells (IRF4), and adipogenic markers (aP2/FABP4, AdipoQ, PPARγ, Leptin, CideA), and immunostaining showed nodular lymphocytic accumulation. Immunohistochemistry staining revealed a higher density of inflammatory cells (CD20, CD3, CD68) in CAH compared to non-CAH myelolipomas. In vitro RNA-sequencing studies using NCI-H295R adrenocortical cells with exogenous exposure to ACTH, testosterone, and 17-hydroxyprogesterone hormones, showed the differential expression of genes involved in cell cycle progression, phosphorylation, and tumorigenesis. Migration of B-lymphocytes was initiated after the hormonal treatment of adrenocortical cells using the Boyden chamber chemotaxis assay, indicating a possible hormonal influence on triggering inflammation and the development of myelolipomas. These findings demonstrate the important role of inflammation and the hormonal milieu in the development of AML in CAH.

Long-term survivors of glioblastoma: Tumor molecular, clinical, and imaging findings.

Background: Glioblastoma (GBM) is the most aggressive primary brain malignancy with <45% living a year beyond diagnosis. Previously published investigations of long-term survivors (LTS) provided clinical data but rarely incorporated a comprehensive clinical and molecular analysis. Herein, we identify clinical, imaging, molecular, and outcome features for 23 GBM-LTS patients and compare them with a matched cohort of short-term survivors (STS). Methods: Molecularly confirmed Isocitrate Dehydrogenase (IDH) wildtype GBM patients living ≥3 years post-diagnosis (NLTS = 23) or <3 years (NSTS = 75) were identified from our Natural History study. Clinical and demographic characteristics were compared. Tumor tissue was analyzed with targeted next generation sequencing (NGS) (NLTS = 23; NSTS = 74) and methylation analysis (NLTS = 18; NSTS = 28). Pre-surgical MRI scans for a subset of LTS (N = 14) and STS control (N = 28) matched on sex, age, and extent of resection were analyzed. Results: LTS tended to be younger. Diagnostic MRIs showed more LTS with T1 tumor hypointensity. LTS tumors were enriched for MGMTp methylation and tumor protein 53 (TP53) mutation. Three patients with classic GBM histology were reclassified based on NGS and methylation testing. Additionally, there were LTS with typical poor prognostic molecular markers. Conclusions: Our findings emphasize that generalized predictions of prognosis are inaccurate for individual patients and underscore the need for complete clinical evaluation including molecular work-up to confirm the diagnosis. Continued accrual of patients to LTS registries that containcomprehensive clinical, imaging, tumor molecular data, and outcomes measures may pro\vide important insights about individual patient prognosis.

Potential prognostic determinants for FET::CREB fusion-positive intracranial mesenchymal tumor.

Intracranial mesenchymal tumor (IMT), FET::CREB fusion-positive is a provisional tumor type in the 2021 WHO classification of central nervous system tumors with limited information available. Herein, we describe five new IMT cases from four females and one male with three harboring an EWSR1::CREM fusion and two featuring an EWSR1::ATF1 fusion. Uniform manifold approximation and projection of DNA methylation array data placed two cases to the methylation class "IMT, subclass B", one to "meningioma-benign" and one to "meningioma-intermediate". A literature review identified 74 cases of IMTs (current five cases included) with a median age of 23 years (range 4-79 years) and a slight female predominance (female/male ratio = 1.55). Among the confirmed fusions, 25 (33.8%) featured an EWSR1::ATF1 fusion, 24 (32.4%) EWSR1::CREB1, 23 (31.1%) EWSR1::CREM, one (1.4%) FUS::CREM, and one (1.4%) EWSR1::CREB3L3. Among 66 patients with follow-up information available (median: 17 months; range: 1-158 months), 26 (39.4%) experienced progression/recurrences (median 10.5 months; range 0-120 months). Ultimately, three patients died of disease, all of whom underwent a subtotal resection for an EWSR1::ATF1 fusion-positive tumor. Outcome analysis revealed subtotal resection as an independent factor associated with a significantly shorter progression free survival (PFS; median: 12 months) compared with gross total resection (median: 60 months; p < 0.001). A younger age (< 14 years) was associated with a shorter PFS (median: 9 months) compared with an older age (median: 49 months; p < 0.05). Infratentorial location was associated with a shorter overall survival compared with supratentorial (p < 0.05). In addition, the EWSR1::ATF1 fusion appeared to be associated with a shorter overall survival compared with the other fusions (p < 0.05). In conclusion, IMT is a locally aggressive tumor with a high recurrence rate. Potential risk factors include subtotal resection, younger age, infratentorial location, and possibly EWSR1::ATF1 fusion. Larger case series are needed to better define prognostic determinants in these tumors.

TGF-ß and BMP signaling are associated with the transformation of glioblastoma to gliosarcoma and then osteosarcoma.

Background: Gliosarcoma, an isocitrate dehydrogenase wildtype (IDH-WT) variant of glioblastoma, is defined by clonal biphasic differentiation into gliomatous and sarcomatous components. While the transformation from a glioblastoma to gliosarcoma is uncommon, the subsequent transformation to osteosarcoma is rare but may provide additional insights into the biology of these typically distinct cancers. We observed a patient initially diagnosed with glioblastoma, that differentiated into gliosarcoma at recurrence, and further evolved to osteosarcoma at the second relapse. Our objective was to characterize the molecular mechanisms of tumor progression associated with this phenotypic transformation. Methods: Tumor samples were collected at all 3 stages of disease and RNA sequencing was performed to capture their transcriptomic profiles. Sequential clonal evolution was confirmed by the maintenance of an identical PTEN mutation throughout the tumor differentiation using the TSO500 gene panel. Publicly available datasets and the Nanostring nCounter technology were used to validate the results. Results: The glioblastoma tumor from this patient possessed mixed features of all 3 TCGA-defined transcriptomic subtypes of an IDH-WT glioblastoma and a proportion of osteosarcoma signatures were upregulated in the original tumor. Analysis showed that enhanced transforming growth factor-ß (TGF-ß) and bone morphogenic protein signaling was associated with tumor transformation. Regulatory network analysis revealed that TGF-ß family signaling committed the lineage tumor to osteogenesis by stimulating the expression of runt-related transcription factor 2 (RUNX2), a master regulator of bone formation. Conclusions: This unusual clinical case provided an opportunity to explore the modulators of longitudinal sarcomatous transformation, potentially uncovering markers indicating predisposition to this change and identification of novel therapeutic targets.

Assessment of The Utility of The Sarcoma DNA Methylation Classifier In Surgical Pathology.

Diagnostic classification of soft tissue tumors is based on histology, immunohistochemistry, genetic findings, and radiologic and clinical correlations. Recently, a sarcoma DNA methylation classifier was developed, covering 62 soft tissue and bone tumor entities. The classifier is based on large-scale analysis of methylation sites across the genome. It includes DNA copy number analysis and determines O 6 methylguanine DNA methyl-transferase methylation status. In this study, we evaluated 619 well-studied soft tissue and bone tumors with the sarcoma classifier. Problem cases and typical examples of different entities were included. The classifier had high sensitivity and specificity for fusion sarcomas: Ewing, synovial, CIC -rearranged, and BCOR -rearranged. It also performed well for leiomyosarcoma, malignant peripheral nerve sheath tumors (MPNST), and malignant vascular tumors. There was low sensitivity for diagnoses of desmoid fibromatosis, neurofibroma, and schwannoma. Low specificity of matches was observed for angiomatoid fibrous histiocytoma, inflammatory myofibroblastic tumor, Langerhans histiocytosis, schwannoma, undifferentiated sarcoma, and well-differentiated/dedifferentiated liposarcoma. Diagnosis of lipomatous tumors was greatly assisted by the detection of MDM2 amplification and RB1 loss in the copy plot. The classifier helped to establish diagnoses for KIT-negative gastrointestinal stromal tumors, MPNSTs with unusual immunophenotypes, and undifferentiated melanomas. O 6 methylguanine DNA methyl-transferase methylation was infrequent and most common in melanomas (35%), MPNSTs (11%), and undifferentiated sarcomas (11%). The Sarcoma Methylation Classifier will likely evolve with the addition of new entities and refinement of the present methylation classes. The classifier may also help to define new entities and give new insight into the interrelationships of sarcomas.

Diffuse Pediatric-type High-grade Glioma Arising in an Ovarian Mature Cystic Teratoma.

Immature neuroectodermal tissue can be found in the ovary as part of an immature teratoma or as part of a teratoma with malignant neuroectodermal transformation. Such lesions may closely resemble central nervous system tumors, but their biologic similarity is unclear. We describe an 18-yr-old female who presented with abdominal pain caused by an ovarian mass with widespread metastases. Histology showed a primitive, high-grade tumor arising in the background of a mature teratoma. The tumor was SOX10 positive, with focal expression of GFAP, S100, NSE, and synaptophysin. Molecular analysis demonstrated co-amplification of PDGFRA and KIT , alterations common in high-grade gliomas. By whole-genome methylation profiling, it clustered into the "diffuse pediatric-type high-grade glioma, RTK1 subtype, subclass c" group. Despite progressing through 2 lines of chemotherapy with widespread metastatic disease, she achieved an excellent response to chemotherapy directed toward aggressive germ cell tumors. This case emphasizes the importance of immunohistochemical, genomic, and epigenetic analyses to accurately classify these exceedingly rare tumors and determine the optimal therapy.

A case of disseminated spinal astroblastoma harboring a MAMLD1::BEND2 fusion.

Astroblastoma, MN1-altered, is a rare neoplasm of the central nervous system (CNS). This malignancy shares similar histopathological features with other CNS tumors, including ependymomas, making it challenging to diagnose. DNA methylation profiling is a new and robust technique that may be used to overcome this diagnostic hurdle. We report the case of a now 25-year-old female diagnosed with what was initially called an ependymoma located in the cervical spine at the age of 2 years old. After initial resection, the tumor recurred multiple times and within 2 years of diagnosis had disseminated disease throughout the brain and spinal cord. She has now undergone over two decades of treatment, including multiple surgical resections, radiation therapy, and administration of numerous chemotherapeutic agents. In 2021, the patient presented to our institution with lumbosacral radicular symptoms due to enlarging lesions within the lumbosacral spine. Reexamination of formalin-fixed, paraffin-embedded material from the patient's tumor using genomic DNA methylation profiling resulted in a diagnostic change from grade III anaplastic ependymoma to astroblastoma, MN1-altered. This work describes another confirmed case of astroblastoma, MN1-altered, to the growing body of literature.