Immune landscape of oncohistone-mutant gliomas reveals diverse myeloid populations and tumor-promoting function.

Histone H3-mutant gliomas are deadly brain tumors characterized by a dysregulated epigenome and stalled differentiation. In contrast to the extensive datasets available on tumor cells, limited information exists on their tumor microenvironment (TME), particularly the immune infiltrate. Here, we characterize the immune TME of H3.3K27M and G34R/V-mutant gliomas, and multiple H3.3K27M mouse models, using transcriptomic, proteomic and spatial single-cell approaches. Resolution of immune lineages indicates high infiltration of H3-mutant gliomas with diverse myeloid populations, high-level expression of immune checkpoint markers, and scarce lymphoid cells, findings uniformly reproduced in all H3.3K27M mouse models tested. We show these myeloid populations communicate with H3-mutant cells, mediating immunosuppression and sustaining tumor formation and maintenance. Dual inhibition of myeloid cells and immune checkpoint pathways show significant therapeutic benefits in pre-clinical syngeneic mouse models. Our findings provide a valuable characterization of the TME of oncohistone-mutant gliomas, and insight into the means for modulating the myeloid infiltrate for the benefit of patients.

CASCADES, a novel SOX2 super-enhancer-associated long noncoding RNA, regulates cancer stem cell specification and differentiation in glioblastoma.

Glioblastoma is the most common primary malignant brain tumor in adults, with a median survival of just over 1 year. The failure of available treatments to achieve remission in patients with glioblastoma (GBM) has been attributed to the presence of cancer stem cells (CSCs), which are thought to play a central role in tumor development and progression and serve as a treatment-resistant cell repository capable of driving tumor recurrence. In fact, the property of "stemness" itself may be responsible for treatment resistance. In this study, we identify a novel long noncoding RNA (lncRNA), cancer stem cell-associated distal enhancer of SOX2 (CASCADES), that functions as an epigenetic regulator in glioma CSCs (GSCs). CASCADES is expressed in isocitrate dehydrogenase (IDH)-wild-type GBM and is significantly enriched in GSCs. Knockdown of CASCADES in GSCs results in differentiation towards a neuronal lineage in a cell- and cancer-specific manner. Bioinformatics analysis reveals that CASCADES functions as a super-enhancer-associated lncRNA epigenetic regulator of SOX2. Our findings identify CASCADES as a critical regulator of stemness in GSCs that represents a novel epigenetic and therapeutic target for disrupting the CSC compartment in glioblastoma.

PPM1G dephosphorylates eIF4E in control of mRNA translation and cell proliferation.

The mRNA 5'cap-binding eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in the control of mRNA translation in health and disease. One mechanism of regulation of eIF4E activity is via phosphorylation of eIF4E by MNK kinases, which promotes the translation of a subset of mRNAs encoding pro-tumorigenic proteins. Work on eIF4E phosphatases has been paltry. Here, we show that PPM1G is the phosphatase that dephosphorylates eIF4E. We describe the eIF4E-binding motif in PPM1G that is similar to 4E-binding proteins (4E-BPs). We demonstrate that PPM1G inhibits cell proliferation by targeting phospho-eIF4E-dependent mRNA translation.

Comparative analysis of methods to reduce activation signature gene expression in PBMCs.

Preserving the in vivo cell transcriptome is essential for accurate profiling, yet factors during cell isolation including time ex vivo and temperature induce artifactual gene expression, particularly in stress-responsive immune cells. In this study, we investigated two methods to mitigate ex vivo activation signature gene (ASG) expression in peripheral blood mononuclear cells (PBMCs): transcription and translation inhibitors (TTis) and cold temperatures during isolation. Comparative analysis of PBMCs isolated with TTis revealed reduced ASG expression. However, TTi treatment impaired responsiveness to LPS stimulation in subsequent in vitro experiments. In contrast, cold isolation methods also prevented ASG expression; up to a point where the addition of TTis during cold isolation offered minimal additional advantage. These findings highlight the importance of considering the advantages and drawbacks of different isolation methods to ensure accurate interpretation of PBMC transcriptomic profiles.

mTOR Pathway Somatic Pathogenic Variants in Focal Malformations of Cortical Development: Novel Variants, Topographic Mapping, and Clinical Outcomes.

Background and Objectives: Somatic and germline pathogenic variants in genes of the mammalian target of rapamycin (mTOR) signaling pathway are a common mechanism underlying a subset of focal malformations of cortical development (FMCDs) referred to as mTORopathies, which include focal cortical dysplasia (FCD) type II, subtypes of polymicrogyria, and hemimegalencephaly. Our objective is to screen resected FMCD specimens with mTORopathy features on histology for causal somatic variants in mTOR pathway genes, describe novel pathogenic variants, and examine the variant distribution in relation to neuroimaging, histopathologic classification, and clinical outcomes. Methods: We performed ultra-deep sequencing using a custom HaloPlexHS Target Enrichment kit in DNA from 21 resected fresh-frozen histologically confirmed FCD type II, tuberous sclerosis complex, or hemimegalencephaly specimens. We mapped the variant alternative allele frequency (AAF) across the resected brain using targeted ultra-deep sequencing in multiple formalin-fixed paraffin-embedded tissue blocks. We also functionally validated 2 candidate somatic MTOR variants and performed targeted RNA sequencing to validate a splicing defect associated with a novel DEPDC5 variant. Results: We identified causal mTOR pathway gene variants in 66.7% (14/21) of patients, of which 13 were somatic with AAF ranging between 0.6% and 12.0%. Moreover, the AAF did not predict balloon cell presence. Favorable seizure outcomes were associated with genetically clear resection borders. Individuals in whom a causal somatic variant was undetected had excellent postsurgical outcomes. In addition, we demonstrate pathogenicity of the novel c.4373_4375dupATG and candidate c.7499T>A MTOR variants in vitro. We also identified a novel germline aberrant splice site variant in DEPDC5 (c.2802-1G>C). Discussion: The AAF of somatic pathogenic variants correlated with the topographic distribution, histopathology, and postsurgical outcomes. Moreover, cortical regions with absent histologic FCD features had negligible or undetectable pathogenic variant loads. By contrast, specimens with frank histologic abnormalities had detectable pathogenic variant loads, which raises important questions as to whether there is a tolerable variant threshold and whether surgical margins should be clean, as performed in tumor resections. In addition, we describe 2 novel pathogenic variants, expanding the mTORopathy genetic spectrum. Although most pathogenic somatic variants are located at mutation hotspots, screening the full-coding gene sequence remains necessary in a subset of patients.

Myositis with prominent B cell aggregates may meet classification criteria for sporadic inclusion body myositis.

The objective of this study was to report the clinical, serological and pathological features of patients with autoimmune myositis other than dermatomyositis, who displayed both muscle weakness on physical examination and prominent B cell aggregates on muscle pathology, defined as ≥ 30 CD20+ cells/aggregate. Specifically, the presence of a brachio-cervical inflammatory myopathies or a sporadic inclusion body myositis (sIBM) phenotype was recorded. Over a three-year period, eight patients were identified from two university neuropathology referral centers. Seven of 8 (88%) patients had an associated connective tissue disease (CTD): rheumatoid arthritis (n=3), systemic sclerosis (n=2), Sjögren's syndrome (n=1) and systemic lupus erythematosus (n=1), while one patient died on initial presentation without a complete serological and cancer investigation. A brachio-cervical phenotype, i.e. neck weakness, proximal weakness more than distal and shoulder abduction weakness greater than hip flexors, was seen in two patients (25%), while one patient had both proximal and diaphragmatic weakness. In contrast, an IBM-like clinical phenotype was seen in the last five patients (63%), who either had finger flexor weakness and/or quadriceps weakness ≤ 4 on the manual muscle testing MRC-5 scale. Although these 5 patients met at least one set of classification criteria for sIBM, an integrated clinico-sero-pathological approach argued against a diagnosis of sIBM. In summary, in a weak patient with myositis plus an associated CTD and lymphoid aggregates at muscle pathology, B cell predominant aggregates may represent a morphological biomarker against a diagnosis of sIBM.

IgG4-Related Disease of the Central Nervous System: A Case Series.

IgG4-related disease (IgG4-RD) is a rare and often misdiagnosed disorder with limited literature that highlights the different neurological presentations of this treatable disease. The diagnosis of IgG4-RD could be challenging, while imaging is fundamental for the diagnosis, biopsy is considered the gold standard. Most cases respond well to steroids and immunosuppressive therapy. This is a case series study that illustrates the varied neurological presentations of IgG4-RD through three different patients that were followed at the Montreal Neurological Institute. This paper takes you through the diagnostic strategy that we followed to accurately diagnose and treat those patients.

CAPTURE ALS: the comprehensive analysis platform to understand, remedy and eliminate ALS.

The absence of disease modifying treatments for amyotrophic lateral sclerosis (ALS) is in large part a consequence of its complexity and heterogeneity. Deep clinical and biological phenotyping of people living with ALS would assist in the development of effective treatments and target specific biomarkers to monitor disease progression and inform on treatment efficacy. The objective of this paper is to present the Comprehensive Analysis Platform To Understand Remedy and Eliminate ALS (CAPTURE ALS), an open and translational platform for the scientific community currently in development. CAPTURE ALS is a Canadian-based platform designed to include participants' voices in its development and through execution. Standardized methods will be used to longitudinally characterize ALS patients and healthy controls through deep clinical phenotyping, neuroimaging, neurocognitive and speech assessments, genotyping and multisource biospecimen collection. This effort plugs into complementary Canadian and international initiatives to share common resources. Here, we describe in detail the infrastructure, operating procedures, and long-term vision of CAPTURE ALS to facilitate and accelerate translational ALS research in Canada and beyond.

Consent Codes: Maintaining Consent in an Ever-expanding Open Science Ecosystem.

We previously proposed a structure for recording consent-based data use 'categories' and 'requirements' - Consent Codes - with a view to supporting maximum use and integration of genomic research datasets, and reducing uncertainty about permissible re-use of shared data. Here we discuss clarifications and subsequent updates to the Consent Codes (v4) based on new areas of application (e.g., the neurosciences, biobanking, H3Africa), policy developments (e.g., return of research results), and further practical considerations, including developments in automated approaches to consent management.

Detection and genomic analysis of BRAF fusions in Juvenile Pilocytic Astrocytoma through the combination and integration of multi-omic data.

BACKGROUND: Juvenile Pilocytic Astrocytomas (JPAs) are one of the most common pediatric brain tumors, and they are driven by aberrant activation of the mitogen-activated protein kinase (MAPK) signaling pathway. RAF-fusions are the most common genetic alterations identified in JPAs, with the prototypical KIAA1549-BRAF fusion leading to loss of BRAF's auto-inhibitory domain and subsequent constitutive kinase activation. JPAs are highly vascular and show pervasive immune infiltration, which can lead to low tumor cell purity in clinical samples. This can result in gene fusions that are difficult to detect with conventional omics approaches including RNA-Seq. METHODS: To this effect, we applied RNA-Seq as well as linked-read whole-genome sequencing and in situ Hi-C as new approaches to detect and characterize low-frequency gene fusions at the genomic, transcriptomic and spatial level. RESULTS: Integration of these datasets allowed the identification and detailed characterization of two novel BRAF fusion partners, PTPRZ1 and TOP2B, in addition to the canonical fusion with partner KIAA1549. Additionally, our Hi-C datasets enabled investigations of 3D genome architecture in JPAs which showed a high level of correlation in 3D compartment annotations between JPAs compared to other pediatric tumors, and high similarity to normal adult astrocytes. We detected interactions between BRAF and its fusion partners exclusively in tumor samples containing BRAF fusions. CONCLUSIONS: We demonstrate the power of integrating multi-omic datasets to identify low frequency fusions and characterize the JPA genome at high resolution. We suggest that linked-reads and Hi-C could be used in clinic for the detection and characterization of JPAs.

An integrated virtual pathology education platform developed using Microsoft Power Apps and Microsoft Teams.

The transition towards digital pathology and an extensive selection of video conferencing platforms have helped provide continuity to education even during the COVID-19 pandemic. Innovative approaches for pathology education, will likely persist beyond the pandemic, as they have powerful didactic potential. While there is a wide selection of software for use as educational tools, an environment to access all resources with ease is clearly lacking. In this technical note, we highlight our customized educational applications built using a low-code approach. Our applications, developed with Microsoft Power Apps, serve both educational and examination purposes and are launched using Microsoft Teams. Building applications using a low-code approach has made our applications very specific to our use and enabled daily distanced education. Combined with existing features on Teams, such as file sharing, meeting scheduling, and messaging, the applications serve as a unique and customizable pathology educational platform.

Generation of patient-derived pluripotent stem cell-lines and CRISPR modified isogenic controls with mutations in the Parkinson's associated GBA gene.

The GBA gene encodes the lysosomal enzyme glucocerebrosidase (GCase), responsible for the hydrolysis of glucocerebroside to glucose and ceramide. Heterozygous GBA mutations have been associated with the development of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We generated two induced pluripotent stem cell (iPSC) lines from PD patients carrying heterozygous GBA W378G or N370S mutations and subsequently produced isogenic control lines using CRISPR/Cas9 genome editing. The patient-derived iPSCs and isogenic control lines maintained full pluripotency, normal karyotypes, and differentiation capacity. All iPSC lines could be differentiated into dopaminergic neurons, thus providing valuable tools for studying PD pathogenesis.

Detection of temozolomide-induced hypermutation and response to PD-1 checkpoint inhibitor in recurrent glioblastoma.

Background: Despite aggressive upfront treatment in glioblastoma (GBM), recurrence remains inevitable for most patients. Accumulating evidence has identified hypermutation induced by temozolomide (TMZ) as an emerging subtype of recurrent GBM. However, its biological and therapeutic significance has yet to be described. Methods: We combined GBM patient and derive GBM stem cells (GSCs) from tumors following TMZ to explore response of hypermutant and non-hypermutant emergent phenotypes and explore the immune relevance of hypermutant and non-hypermutant states in vivo. Results: Hypermutation emerges as one of two possible mutational subtypes following TMZ treatment in vivo and demonstrates distinct phenotypic features compared to non-hypermutant recurrent GBM. Hypermutant tumors elicited robust immune rejection in subcutaneous contexts which was accompanied by increased immune cell infiltration. In contrast, immune rejection of hypermutant tumors were stunted in orthotopic settings where we observe limited immune infiltration. Use of anti-PD-1 immunotherapy showed that immunosuppression in orthotopic contexts was independent from the PD-1/PD-L1 axis. Finally, we demonstrate that mutational burden can be estimated from DNA contained in extracellular vesicles (EVs). Conclusion: Hypermutation post-TMZ are phenotypically distinct from non-hypermutant GBM and requires personalization for appropriate treatment. The brain microenvironment may be immunosuppressive and exploration of the mechanisms behind this may be key to improving immunotherapy response in this subtype of recurrent GBM.

Capillary pathology with prominent basement membrane reduplication is the hallmark histopathological feature of scleromyositis.

AIMS: We aim to perform ultrastructural and histopathological analysis of muscle biopsies from a large group of systemic sclerosis (SSc) patients, including some with early/mild SSc features, and examine whether capillary pathology differentiates 'scleromyositis' (SM) from other auto-immune myositis (AIM) subsets. METHODS: Muscle biopsies from a total of 60 SM patients and 43 AIM controls from two independent cohorts were examined by electron microscopy, collagen-4 immunofluorescence (Col4IF) and routine light microscopy. RESULTS: Ultrastructural examination revealed prominent capillary basement membrane (BM) reduplication (4+ layers in >50% of capillaries) in 65% of SM vs 0% of AIM controls (p < 0.001). In SM cases without prominent BM reduplication, capillary dilation was the most distinctive feature, present in 8% of capillaries in SM vs 2% in controls (p = 0.001). Accumulation of ensheathed pericyte processes was another characteristic feature of SM and closely correlated with the degree of BM reduplication (r = 0.833, p < 0.001). On light microscopy, BM marker Col4IF revealed more frequent capillary enlargement in SM than in controls (84% vs 21%, p < 0.001). SM cases were classified as non-inflammatory myopathy (36%), non-specific myositis (33%) or immune-mediated necrotizing myopathy (31%), but despite this histopathological heterogeneity, prominent BM reduplication remained a constant finding. In the 16 SM patients with early/mild SSc features, 63% showed prominent BM reduplication. CONCLUSIONS: These results show that capillary pathology, and in particular prominent capillary BM reduplication, is the hallmark histopathological feature of SM even in patients with early/mild SSc and support the concept of SM as an organ manifestation of SSc and a distinct subset of AIM.

Novel homozygous nonsense mutation of MLIP and compensatory alternative splicing.

Despite the growing accessibility of clinical sequencing, functional interpretation of variants remains a major hurdle to molecular diagnostics of Mendelian diseases. We aimed to describe a new adult-onset myopathy with muscle weakness and hyperCKemia caused by a nonsense variant in muscular LMNA-interacting protein (MLIP). Following RNA-sequencing, differential expression analysis uncovered a significant downregulation of this gene, which had a surprisingly mild effect on MLIP protein expression. RT-PCR and long-read sequencing (LRS) both support an important transcriptome shift in the patient, where decreased MLIP levels are seemingly due to nonsense-mediated decay of transcripts containing the exon 5 mutation. Moreover, a compensatory mechanism upregulates the functionally lacking isoforms and generates novel transcripts. These results support the recently discovered clinical implications of MLIP variants in myopathies, highlighting for the first time its relevance in adult-onset cases. These results also underline the power of LRS as a tool for the functional assessment of variants of unknown significance (VUS), as well as the definition of accurate isoform profile annotations in a tissue-specific manner.

Generation of homozygous PRKN, PINK1 and double PINK1/PRKN knockout cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing.

Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinson's disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinson's disease.

Myositis with prominent B-cell aggregates causing shrinking lung syndrome in systemic lupus erythematosus: a case report.

BACKGROUND: Shrinking lung syndrome (SLS) is a rare manifestation of systemic lupus erythematosus (SLE) characterized by decreased lung volumes and diaphragmatic weakness in a dyspneic patient. Chest wall dysfunction secondary to pleuritis is the most commonly proposed cause. In this case report, we highlight a new potential mechanism of SLS in SLE, namely diaphragmatic weakness associated with myositis with CD20 positive B-cell aggregates. CASE PRESENTATION: A 51-year-old Caucasian woman was diagnosed with SLE and secondary Sjögren's syndrome based on a history of pleuritis, constrictive pericarditis, polyarthritis, photosensitivity, alopecia, oral ulcers, xerophthalmia and xerostomia. Serologies were significant for positive antinuclear antibodies, anti-SSA, lupus anticoagulant and anti-cardiolopin. Blood work revealed a low C3 and C4, lymphopenia and thrombocytopenia. She was treated with with low-dose prednisone and remained in remission with oral hydroxychloroquine. Seven years later, she developed mild proximal muscle weakness and exertional dyspnea. Pulmonary function testing revealed a restrictive pattern with small lung volumes. Pulmonary imaging showed elevation of the right hemidiaphragm without evidence of interstitial lung disease. Diaphragmatic ultrasound was suggestive of profound diaphragmatic weakness and dysfunction. Based on these findings, a diagnosis of SLS was made. Her proximal muscle weakness was investigated, and creatine kinase (CK) levels were normal. Electromyography revealed fibrillation potentials in the biceps, iliopsoas, cervical and thoracic paraspinal muscles, and complex repetitive discharges in cervical paraspinal muscles. Biceps muscle biopsy revealed dense endomysial lymphocytic aggregates rich in CD20 positive B cells, perimysial fragmentation with plasma cell-rich perivascular infiltrates, diffuse sarcolemmal upregulation of class I MHC, perifascicular upregulation of class II MHC, and focal sarcolemmal deposition of C5b-9. Treatment with prednisone 15 mg/day and oral mycophenolate mofetil 2 g/day was initiated. Shortness of breath and proximal muscle weakness improved significantly. CONCLUSION: Diaphragmatic weakness was the inaugural manifestation of myositis in this patient with SLE. The spectrum of myologic manifestations of myositis with prominent CD20 positive B-cell aggregates in SLE now includes normal CK levels and diaphragmatic involvement, in association with SLS.