The Role of Mesenchymal Reprogramming in Malignant Clonal Evolution and Intra-Tumoral Heterogeneity in Glioblastoma.

Glioblastoma (GBM) is the most common yet uniformly fatal adult brain cancer. Intra-tumoral molecular and cellular heterogeneities are major contributory factors to therapeutic refractoriness and futility in GBM. Molecular heterogeneity is represented through molecular subtype clusters whereby the proneural (PN) subtype is associated with significantly increased long-term survival compared to the highly resistant mesenchymal (MES) subtype. Furthermore, it is universally recognized that a small subset of GBM cells known as GBM stem cells (GSCs) serve as reservoirs for tumor recurrence and progression. The clonal evolution of GSC molecular subtypes in response to therapy drives intra-tumoral heterogeneity and remains a critical determinant of GBM outcomes. In particular, the intra-tumoral MES reprogramming of GSCs using current GBM therapies has emerged as a leading hypothesis for therapeutic refractoriness. Preventing the intra-tumoral divergent evolution of GBM toward the MES subtype via new treatments would dramatically improve long-term survival for GBM patients and have a significant impact on GBM outcomes. In this review, we examine the challenges of the role of MES reprogramming in the malignant clonal evolution of glioblastoma and provide future perspectives for addressing the unmet therapeutic need to overcome resistance in GBM.

Spatial transcriptomics analysis identifies a tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease.

Leptomeningeal disease (LMD) remains a rapidly lethal complication for late-stage melanoma patients. Here, we characterize the tumor microenvironment of LMD and patient-matched extra-cranial metastases using spatial transcriptomics in a small number of clinical specimens (nine tissues from two patients) with extensive in vitro and in vivo validation. The spatial landscape of melanoma LMD is characterized by a lack of immune infiltration and instead exhibits a higher level of stromal involvement. The tumor-stroma interactions at the leptomeninges activate tumor-promoting signaling, mediated through upregulation of SERPINA3. The meningeal stroma is required for melanoma cells to survive in the cerebrospinal fluid (CSF) and promotes MAPK inhibitor resistance. Knocking down SERPINA3 or inhibiting the downstream IGR1R/PI3K/AKT axis results in tumor cell death and re-sensitization to MAPK-targeting therapy. Our data provide a spatial atlas of melanoma LMD, identify the tumor-promoting role of meningeal stroma, and demonstrate a mechanism for overcoming microenvironment-mediated drug resistance in LMD.

Training simulated participants for role portrayal and feedback practices in communication skills training: A BEME scoping review: BEME Guide No. 86.

BACKGROUND: Providing feedback is a key aspect of simulated participants' (SPs) educational work. In teaching contexts, the ability to provide feedback to learners is central to their role. Suboptimal feedback practices may deny learners the valuable feedback they need to learn and improve. This scoping review systematically maps the evidence related to SPs' role as educators and identifies how SPs prepare for their role and feedback practices. METHODS: The authors conducted a scoping review and included a group of international stakeholders with experience and expertise in SP methodology. Five online databases were systematically searched and ERIC, MedEdPortal and MedEdPublish were hand searched to identify relevant studies. Inclusion/exclusion criteria were developed. Data screening and subsequently data charting were performed in pairs. The results of data charting were thematically analysed including categories relating to the Association of SP Educators (ASPE) Standards of Best Practice (SOBP). RESULTS: From 8179 articles identified for the title and abstract screening, 98 studies were included. Studies reported the benefit of SPs' authentic role portrayal and feedback interactions for learners and on the reported learning outcomes. Data was heterogeneous with a notable lack of consistency in the detail regarding the scenario formats for communication skills training interventions, SP characteristics, and approaches to training for feedback and role portrayal. CONCLUSIONS: The published literature has considerable heterogeneity in reporting how SPs are prepared for role portrayal and feedback interactions. Additionally, our work has identified gaps in the implementation of the ASPE SOBP, which promotes effective SP-learner feedback interactions. Further research is required to identify effective applications of SP methodology to prepare SPs for their role as educators.

Development of a "Geo-Tagged" tumor sample registry: intra-operative linkage of sample location to imaging.

PURPOSE: There is a growing body of literature documenting glioma heterogeneity in terms of radiographic, histologic, molecular, and genetic characteristics. Incomplete spatial specification of intraoperative tumor samples may contribute to variability in the results of pathological and biological investigations. We have developed a system, termed geo-tagging, for routine intraoperative linkage of each tumor sample to its location via neuronavigation. METHODS: This is a single-institution, IRB approved, prospective database of undergoing clinically indicated surgery. We evaluated relevant factors affecting data collection by this registry, including tumor and surgical factors (e.g. tumor volume, location, grade and surgeon). RESULTS: Over a 2-year period, 487 patients underwent craniotomy for an intra-axial tumor. Of those, 214 underwent surgery for a newly diagnosed or recurrent glioma. There was significant variation in the average number of samples collected per registered case, with a range of samples from 2.53 to 4.75 per tumor type. Histology and grade impacted on sampling with a range of 2.0 samples per tumor in Grade four, IDH-WT gliomas to 4.5 samples in grade four, IDH-mutant gliomas. The range of cases with sampling per surgeon was 6 to 99 with a mean of 47.6 cases and there was a statistically significant differences between surgeons. Tumor grade did not have a statistically significant impact on number of samples per case. No significant correlation was found between the number of samples collected and enhancing tumor volume, EOR, or volume of tumor resected. CONCLUSION: We are using the results of this analysis to develop a prospective sample collection protocol.

HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis.

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.

Epigenetic Activation of TUSC3 Sensitizes Glioblastoma to Temozolomide Independent of MGMT Promoter Methylation Status.

Temozolomide (TMZ) is an important first-line treatment for glioblastoma (GBM), but there are limitations to TMZ response in terms of durability and dependence on the promoter methylation status of the DNA repair gene O6-methylguanine DNA methyltransferase (MGMT). MGMT-promoter-hypermethylated (MGMT-M) GBMs are more sensitive to TMZ than MGMT-promoter-hypomethylated (MGMT-UM) GBMs. Moreover, TMZ resistance is inevitable even in TMZ-sensitive MGMT-M GBMs. Hence, epigenetic reprogramming strategies are desperately needed in order to enhance TMZ response in both MGMT-M and MGMT-UM GBMs. In this study, we present novel evidence that the epigenetic reactivation of Tumor Suppressor Candidate 3 (TUSC3) can reprogram sensitivity of GBM stem cells (GSCs) to TMZ irrespective of MGMT promoter methylation status. Interrogation of TCGA patient GBM datasets confirmed TUSC3 promoter regulation of TUSC3 expression and also revealed a strong positive correlation between TUSC3 expression and GBM patient survival. Using a combination of loss-of-function, gain-of-function and rescue studies, we demonstrate that TUSC3 reactivation is associated with enhanced TMZ response in both MGMT-M and MGMT-UM GSCs. Further, we provide novel evidence that the demethylating agent 5-Azacitidine (5-Aza) reactivates TUSC3 expression in MGMT-M GSCs, whereas the combination of 5-Aza and MGMT inhibitor Lomeguatrib is necessary for TUSC3 reactivation in MGMT-UM GSCs. Lastly, we propose a pharmacological epigenetic reactivation strategy involving TUSC3 that leads to significantly prolonged survival in MGMT-M and MGMT-UM orthotopic GSCs models. Collectively, our findings provide a framework and rationale to further explore TUSC3-mediated epigenetic reprogramming strategies that could enhance TMZ sensitivity and outcomes in GBM. Mechanistic and translational evidence gained from such studies could contribute towards optimal design of impactful trials for MGMT-UM GBMs that currently do not have good treatment options.

Characterization of Immune Infiltrates Associated With Radiation Necrosis in the Setting of Brain Metastases Following Stereotactic Radiosurgery and Immunotherapy: A Retrospective Cohort Analysis.

INTRODUCTION: Radiation necrosis (RN) is caused by vascular damage and brain parenchymal injury resulting in inflammation following radiotherapy (RT) for brain metastases. The impact of immunotherapy (IO) on the immune cellular microenvironment in patients' brain metastases is unknown. The objective of this study was to characterize the inflammatory microenvironment in the setting of RN compared to recurrent metastasis and determine whether IO treatment affects the cellular infiltrates. METHODS: Adult patients with brain metastases from solid tumors who received both systemic IO and RT prior to resection of intracranial lesions were retrospectively analyzed. The resection either showed biopsy-proven RN or recurrent tumor. A group of patients who developed RN and were not on IO was reviewed as well. A total of 18 patients were categorized into one of three groups: necrosis, IO+RT; tumor, IO+RT; and necrosis, RT. Surgical specimens were stained for immune and inflammatory components and reviewed by a neuro-pathologist who remained blinded during the analysis. The presence or absence of lymphocytes, perivascular cuffs, plasma cells, macrophages, and fibrinoid vascular changes was characterized in a semiquantitative manner. RESULTS: The median age was 61.5 years (range 37-82 years). Seventy-seven percent of primary cancers were melanoma. Patients with RN were more likely to exhibit immune infiltrates compared to patients with recurrent metastasis. Limited analysis showed 100% of patients in "necrosis, IO+RT" had quantifiable cell counts; conversely, 83.3% of patients in "tumor, IO+RT" lacked quantifiable cell counts. Additionally, 83.3% of patients in "necrosis, RT" showed immune cells, including lymphocytes, macrophages, plasma cells, and cuffing. CONCLUSION: The immune microenvironment of brain metastasis following RT and IO showed higher levels of cell infiltrates in the RN setting versus the recurrent tumor setting. Patients who received prior IO compared to those with no IO had similar immune cell infiltrates adjacent to RN. Lower levels of immune cells in tumor recurrence following IO and RT raise the possibility that an environment lacking primed immune cells may decrease the efficacy of IO.

Novel SOX10 indel mutations drive schwannomas through impaired transactivation of myelination gene programs.

BACKGROUND: Schwannomas are common peripheral nerve sheath tumors that can cause severe morbidity given their stereotypic intracranial and paraspinal locations. Similar to many solid tumors, schwannomas and other nerve sheath tumors are primarily thought to arise due to aberrant hyperactivation of the RAS growth factor signaling pathway. Here, we sought to further define the molecular pathogenesis of schwannomas. METHODS: We performed comprehensive genomic profiling on a cohort of 96 human schwannomas, as well as DNA methylation profiling on a subset. Functional studies including RNA sequencing, chromatin immunoprecipitation-DNA sequencing, electrophoretic mobility shift assay, and luciferase reporter assays were performed in a fetal glial cell model following transduction with wildtype and tumor-derived mutant isoforms of SOX10. RESULTS: We identified that nearly one-third of sporadic schwannomas lack alterations in known nerve sheath tumor genes and instead harbor novel recurrent in-frame insertion/deletion mutations in SOX10, which encodes a transcription factor responsible for controlling Schwann cell differentiation and myelination. SOX10 indel mutations were highly enriched in schwannomas arising from nonvestibular cranial nerves (eg facial, trigeminal, vagus) and were absent from vestibular nerve schwannomas driven by NF2 mutation. Functional studies revealed these SOX10 indel mutations have retained DNA binding capacity but impaired transactivation of glial differentiation and myelination gene programs. CONCLUSIONS: We thus speculate that SOX10 indel mutations drive a unique subtype of schwannomas by impeding proper differentiation of immature Schwann cells.

Epstein­Barr virus­associated primary central nervous system lymphoma in an immunosuppressed patient with a comorbid autoimmune disorder: A case report.

Patients with primary central nervous system lymphoma (PCNSL) typically present with non-focal neurological symptoms, including disorientation, poor balance and memory loss with unifocal or multifocal periventricular lesions seen on MRI. Deviations from these characteristic findings can delay diagnosis and lead to additional diagnostic tests being needed. The present study reports a 68-year-old man with a recent varicella zoster infection and history of acetylcholine receptor antibody-positive myasthenia gravis who received mycophenolate mofetil for 22 years. He presented with left eye vision changes and cognitive memory deficits. A brain MRI showed an enhancing lesion within his left medulla extending to the cerebellum. Cerebrospinal fluid analysis was positive for Epstein-Barr virus (EBV) and negative for malignancy. He was diagnosed with varicella zoster virus vasculopathy. At 3 months later, a repeat brain MRI showed multiple new enhancing lesions developing bilaterally along the periventricular white matter. Soon after, he presented to a local ER with acute left-sided blurry vision and worsening memory loss, and he began receiving steroids. Because of rapid symptom progression, he underwent resection of the left frontal lesion, which showed EBV-induced diffuse large B-cell lymphoma (DLBCL). Mycophenolate mofetil was discontinued, and within 24 h of one dose of intravenous 500 mg/m2 rituximab, he had a dramatic improvement in left eye vision and memory loss. He experienced mixed responses to rituximab after 3 cycles. Following one dose of high-dose methotrexate, he developed subsequent chronic kidney disease and required dialysis. He received whole-brain radiation therapy with craniospinal radiation and is currently in complete remission. An EBV-induced DLBCL diagnosis should be highly considered for patients with periventricular lesions and EBV-positive cerebrospinal fluid. Misdiagnosis or delay in PCNSL diagnosis because of atypical features in disease presentation and radiographic findings could lead to PCNSL progression and worsening neurological deficits.

Spatial transcriptomics analysis identifies a unique tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease.

Leptomeningeal disease (LMD) remains a rapidly lethal complication for late-stage melanoma patients. The inaccessible nature of the disease site and lack of understanding of the biology of this unique metastatic site are major barriers to developing efficacious therapies for patients with melanoma LMD. Here, we characterize the tumor microenvironment of the leptomeningeal tissues and patient-matched extra-cranial metastatic sites using spatial transcriptomic analyses with in vitro and in vivo validation. We show the spatial landscape of melanoma LMD to be characterized by a lack of immune infiltration and instead exhibit a higher level of stromal involvement. We show that the tumor-stroma interactions at the leptomeninges activate pathways implicated in tumor-promoting signaling, mediated through upregulation of SERPINA3 at the tumor-stroma interface. Our functional experiments establish that the meningeal stroma is required for melanoma cells to survive in the CSF environment and that these interactions lead to a lack of MAPK inhibitor sensitivity in the tumor. We show that knocking down SERPINA3 or inhibiting the downstream IGR1R/PI3K/AKT axis results in re-sensitization of the tumor to MAPK-targeting therapy and tumor cell death in the leptomeningeal environment. Our data provides a spatial atlas of melanoma LMD, identifies the tumor-promoting role of meningeal stroma, and demonstrates a mechanism for overcoming microenvironment-mediated drug resistance unique to this metastatic site.