Abstracts of the 100th Annual Meeting June 6–9, 2024 Olympic Valley, California (2024)

Platform Presentations #1 – #64
PLATFORM 1: Neurodegenerative: Alzheimer
1
Autopsy findings versus biomarker outcomes in a clinical trial of anti-Aβ therapies in dominantly inherited Alzheimer disease

Background

Clinical trials of anti-Aβ monoclonal antibodies in Alzheimer disease (AD) infer target engagement from Aβ positron emission tomography (PET) and/or fluid biomarkers such as cerebrospinal fluid (CSF) Aβ42/40. However, these biomarkers measure Aβ deposits indirectly and/or incompletely. In contrast, postmortem neuropathologic assessments allow direct investigation of treatment effects on brain Aβ deposits and on many other pathologic features.

Methods

From a clinical trial of anti-Aβ monoclonal antibodies in dominantly inherited AD, we measured immunohistochemistry area fractions (AFs) for Aβ (10D5), tauopathy (PHF1), microgliosis (IBA1) and astrocytosis (GFAP) in 10 brain regions from 10 trial cases–representing gantenerumab (n = 4), solanezumab (n = 4), and placebo/no-treatment (n = 2) arms–and 10 observational study cases. Gantenerumab, solanezumab, and control (placebo/no-treatment/observational) groups were compared based on these AFs, antemortem Aβ PET (11C-PiB) standardized uptake value ratios (SUVRs), and CSF (Aβ42/40, p-tau181, t-tau) biomarkers. Five controls lacked CSF and PET data.

Results

CSF Aβ42/40 showed significant increase in the gantenerumab arm versus controls; CSF t-tau showed a corroborating decrease; CSF p-tau181 showed no significant difference. Aβ PET SUVRs showed significant decreases in the gantenerumab arm versus controls in temporal cortex, caudate, putamen, and thalamus. Strikingly, after continued gantenerumab administration between Aβ PET and autopsy, Aβ AFs were significantly lower in the gantenerumab arm versus control in frontal, temporal, parietal, and occipital cortices, anterior cingulate, hippocampus, caudate, putamen, thalamus, and cerebellar gray matter; only posterior cingulate and cerebellar white matter comparisons were non-significant. In contrast, AFs of tauopathy, microgliosis, and astrocytosis showed no differences across groups.

Conclusions

Our results suggest that gantenerumab treatment, as administered, reduced Aβ deposits–albeit incompletely–in a dose-dependent manner, without significantly altering tauopathy, microgliosis, or astrocytosis. They also suggest that this partial reversal of AD pathology may be optimized with higher doses, more effective anti-Aβ therapeutics, earlier intervention, and/or combined treatments in future clinical trials.

2
Cognitive impairment in Primary Age-Related Tauopathy: A Community-Based Autopsy Study

Background

Primary age-related tauopathy (PART) is characterized by neurofibrillary tangles in the medial temporal lobe (MTL), especially hippocampus (HIP), with no or sparse amount of amyloid-beta (Aβ) plaques. However, there is evidence that tau pathology in PART might be present outside of the MTL. Given this restricted pathoanatomical pattern, we aimed to study tau-tangles inside and outside MTL to investigate downstream effects on cognition.

Methods

We included 527 neuropathologically confirmed PART participants (Thal 0-II, Braak I-IV; age-at-death=88 years; women=63%) from community-based cohort studies of aging. All participants underwent annual clinical evaluations including detailed cognitive testing. Tau-tangle density in the MTL, specifically HIP and outside of the MTL, specifically the neocortex of the inferior temporal lobe (IT) was evaluated by AT8-immunohistohemistry. Multivariable linear mixed-effects models examined the association between regional tau-tangles with decline in global cognition and 5 cognitive domains after controlling demographics, LATE-NC, Lewy bodies, and vascular pathologies.

Results

Tau-tangle pathology was very commonly present in the HIP (N = 524/527) but also common in the IT (N = 449/527). All participants who had tau-tangles in IT also had tangles in HIP. The mean HIP-tau-tangle and IT-tau-tangle density was 2.28(SD = 1.29) and 0.69(SD = 0.58), respectively. Tangle burden in both regions were separately associated with decline in global cognition (HIP= est=-0.005;SE=0.002,p=0.05 and IT= est=-0.016;SE=0.005,p=0.003) and 2 cognitive domains: episodic and semantic memory. In contrast, when included together in the same model the association of HIP-tangle burden with cognitive decline was attenuated and no longer significant but the association of IT-tangle burden persisted (est=-0.013; SE = 0.006, p = 0.038). These findings remained robust after controlling Aβ.

Conclusions

These results indicate that tangles outside the MTL are associated with cognition in PART individuals, independent of tangles inside MTL. Future studies on tau-tangle burden outside the MTL are needed to advance our understanding of the extent to which age-related tauopathy changes impact cognitive function.

3
Shared Transcriptional Changes in Neurons with Tau Pathology in PART and AD

Background

Tau pathology is common in age-related neurodegenerative diseases. Tau pathology in primary age-related tauopathy (PART) and in Alzheimer’s disease (AD) has a similar biochemical structure and anatomic distribution, which is distinct from tau pathology in other diseases. However, the molecular changes associated with intraneuronal tau pathology in PART and AD, and whether these changes are similar in the two diseases, is largely unexplored.

Methods

Using GeoMx spatial transcriptomics, mRNA was quantified in CA1 pyramidal neurons with tau pathology and adjacent neurons without tau pathology in PART andAD, and compared to control cases without pathology. Transcriptional changes were analyzed for differential gene expression and for coordinated patterns of gene expression associated with both disease state and intraneuronal tau pathology.

Results

Gene expression changes associated with intraneuronal tau pathology were similar in PART and AD. Synaptic gene expression was decreased overall in neurons in AD and PART compared to control cases. However, this decrease was largely driven by neurons lacking tau pathology. Two novel gene expression signatures associated with intraneuronal tau were identified by examining coordinated patterns of gene expression, which were similar in both conditions and shared across excitatory cortical neurons in an independent dataset in AD. Genes in the up-regulated expression pattern were enriched in calcium regulation and synaptic function pathways, specifically in synaptic exocytosis.

Conclusions

PART and AD show similar transcriptional changes associated with intraneuronal tau pathology in hippocampal neurons, raising the possibility of a mechanistic relationship between the tau pathology in the two diseases. Intraneuronal tau pathology was also associated with increased expression of genes associated with synaptic function and calcium regulation compared to tau-negative disease neurons. The findings highlight the power of molecular analysis stratified by pathology in neurodegenerative disease and provide novel insight into common molecular pathways associated with intraneuronal tau in PART and AD.

4
Brain Metal Concentrations and Alzheimer’s Disease Neuropathological Change in Total Joint Arthroplasty Patients at Autopsy

Background

The long-term systemic effects of total joint arthroplasty (TJA) are poorly understood relative to the high prevalence of individuals with TJAs. Recent studies suggest that cognitive decline might be slightly faster amongst older patients with TJAs, however the underlying mechanisms and neuropathological changes associated with TJA remain uncertain. One hypothesis is that components of artificial joints shed metal over time, resulting in systemic spread and accumulation in the nervous system, potentially contributing to the mechanisms of neurodegeneration. Herein, we measured metal concentrations in brain tissue and correlated these findings to Alzheimer’s Disease Neuropathological Change (ADNC) in an autopsy cohort of patients with and without TJA.

Methods

This study used autopsy tissue and specimens from 88 decedents with TJA and 89 matched non-TJA controls. Ultra-trace metal concentrations (aluminum, chromium, cobalt, manganese, molybdenum, nickel, titanium, and vanadium) were measured from frozen occipital lobe tissues via triple-quadrupole inductively coupled plasma mass spectrometry. Alzheimer’s disease neuropathologic change assessment was performed following National Institute on Aging-Alzheimer’s Association criteria. Analyzes employed ordinal logistic regression models.

Results

The results show that in both TJA patients and controls, elevated concentrations of titanium, manganese, cobalt, and molybdenum were associated with increased odd-ratios of elevated ADNC A and C scores. Cobalt and titanium concentrations were found to be significantly elevated in TJA patients relative to controls (control vs TJA, titanium: 38.57 ng/g and 54.11 ng/g, p = 0.023; cobalt: 20.68 and 24.69 ng/g, p = 0.024). The increased titanium and cobalt concentrations in TJA patients, also significantly correlated to higher degrees of amyloid pathology, particularly neuritic plaques, relative to controls.

Conclusions

Systemic accumulation of metal debris in TJA patients is well documented. The study observed higher concentrations of cobalt and titanium in the brain, as well as, higher ADNC A and C scores in those with higher metal concentrations.

5
Clinical and Pathological Correlates of ARTAG in the NACC Neuropathology Data Set

Background

The pathoetiology of age-related tau astrogliopathy (ARTAG) is poorly understood. Insights can be gained by analyzing risk factors and comorbid pathologies. Here we addressed the question of which prevalent co-pathologies are observed with increased frequency in brains with ARTAG.

Methods

The study sample was the National Alzheimer’s Coordinating Center (NACC) data set. Data from persons with unusual conditions (e.g. frontotemporal dementia) were excluded leaving 504 individual autopsied research participants, clustering from 20 different ADRCs, autopsied since 2020; ARTAG was reported in 222 (44.0%) of included participants.

Results

As has been shown previously, ARTAG was increasingly frequent with older age and in males. The presence and severity of other common subtypes of pathology that were previously linked to dementia were analyzed, stratifying for the presence of ARTAG. ARTAG was relatively more likely to be found in brains with limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC), and in brains with comorbid cerebrovascular pathology (arteriolosclerosis and/or brain infarcts). However, ARTAG was not associated with severe Alzheimer’s disease neuropathologic change (ADNC), or primary age-related tauopathy (PART). In a subset analysis of 167 participants with neurocognitive testing data, there was a trend for ARTAG pathology to be associated with cognitive impairment as assessed with MMSE scores (P = 0.07). A limitation of the study was that there were missing data about ARTAG pathologies, with incomplete operationalization of ARTAG according to anatomic region and pathologic.

Conclusions

Overall, ARTAG was not associated with ADNC or PART, whereas prior observations about ARTAG occurring with increased frequency in aging, males, and brains with LATE-NC were replicated. It remains to be determined whether the increased frequency of ARTAG in brains with comorbid cerebrovascular pathology is related to local infarctions or neuroinflammatory signaling, or with some other set of correlated factors including blood-brain barrier dysfunction.

6
Environmental-Pathology Interactions in Neurodegenerative Disease Dementia

Background

Environmental factors, in particular, neighborhood socioeconomic status and air pollution are linked to cognitive dysfunction. Whether the association relates to neuropathologic changes is uncertain.

Methods

In a cross-sectional study with 419 autopsy cases collected from 2011 to 2023, we examined relationships among neighborhood disadvantage measured by categorized Area Deprivation Index (ADI) national rankings, cognitive measures, and 10 dementia-associated proteinopathies and cerebrovascular disease (Thal amyloid phase, Braak stage, CERAD score, Alzheimer’s disease neuropathological change (ADNC), Lewy body disease (LBD), limbic-predominant age related TDP-encephalopathy (LATE), large infarcts, amyloid angiopathy, arteriolosclerosis, and cerebrovascular disease in relation to vascular cognitive impairment (VCING)). In a subsequent cross-sectional study with 622 cases from 2000 to 2016, we explored relationships among annual average exposure to fine particulate matter (PM2.5) air pollution estimated at the geocoded residential address for each case for year of death, cognitive measures, and 10 pathologies. All analyses were performed with adjusted regression models.

Results

Higher ADI and PM2.5 concentrations were associated with greater cognitive impairment as measured by Mini-Mental State Examination (MMSE) or Clinical Dementia Rating Sum of Boxes (CDR-SB). While ADI was not associated with an increase in dementia-associated neuropathologic changes, exposure to higher PM2.5 was associated with overall higher neuropathologic burden in major dementia-associated proteinopathies and cerebrovascular disease including higher ADNC level, LBD stage, and VCING level. Finally, the significant association between ADI and cognition was independent of overall neuropathologic changes, while the association between PM2.5 and cognition was largely mediated by increasing ADNC level.

Conclusions

Environmental factors appear to increase risk of dementia through differential pathways. Neighborhood disadvantage appears to reduce cognitive reserve independent of neuropathologic changes, while air pollution appears to affect brain maintenance resulting in exacerbation of ADNC.

7
Early Alzheimer’s Disease-Like Neuropathology in the Nonhuman Primate Chlorocebus Aethiops: Relationships with Age and Physical Function

Background

Nonhuman primates are important translational models of human disease due to their conserved genomic sequence, humanoid anatomy, and susceptibility to similar diseases, including cerebral beta amyloidosis. Specifically, Caribbean vervets (Chlorocebus aethiops sabaeus), may serve as an excellent model for AD. We assessed the brains of 18 deeply phenotyped Caribbean vervets for AD-like neuropathology to further evaluate their utility as a model for early AD-like pathology and to investigate the relationships between plaque burden and other AD-risk phenotypes.

Methods

We performed neuropathologic assessments on 18 vervets. CSF was collected and gait speed was measured in 8 of these. At necropsy, we evaluated 23 brain regions for both Aβ and phospho-tau pathology. Plaque burden was assessed and heat maps were generated to show the distribution across the brain. Gait speed was defined as the average of 5 observations of the time it took to walk between two points. The last gait speed prior to death was used in analysis. Statistical analyses were performed to assess the relationships between AD-like neuropathology, age, and gait speed.

Results

Aβ plaques were identified 10 of the 18 animals. A linear regression model demonstrated a direct relationship between age and total plaque counts (p value = 0.012). Average gait speed was significantly slower in animals with beta amyloid plaques compared to those without (p = 0.05). Linear regression demonstrated an inverse relationship between total plaque burden and average gait speed (p value = 0.034). Analyses of relationships between plaque load, and fluid biomarkers and cognition are underway.

Conclusions

These results support that Caribbean vervets serve as an excellent model for early AD-like neuropathology. In additional to developing Aβ plaques within the cerebrum in a pattern similar to humans, we have demonstrated a direct correlation with age and plaque burden as well as an inverse relationship between plaque burden and average gait speed.

8
Machine Learning Analysis of Amyloid-β Pathologies and Their Correlations in 131 Cases from an Alzheimer’s Disease Research Center

Background

Accurate and scalable quantification of pathologies is crucial for deeper disease phenotyping and furthering research, especially in Alzheimer Disease (AD). This multidisciplinary study addresses the current limitations on neuropathology by leveraging a Machine Learning (ML) pipeline to perform a granular quantification of Amyloid-β (Aβ) deposits and assessing their distribution in the temporal lobe.

Methods

Utilizing available 131 whole-slide-imaging from consecutive autopsied cases at the UC Davis Alzheimer Disease Research Center, our objectives were threefold: 1) Validate an automatic workflow for Aβ deposit quantification in White Matter (WM) and Grey Matter (GM); 2) define the distributions of different Aβ deposit types, and 3) investigate clinicopathological correlates of Aβ deposits with dementia status defined by prior clinical evaluation, and the presence of mixed pathology.

Results

Our methodology highlights the robustness and efficacy of the ML pipeline, demonstrating proficiency akin to experts’ evaluations. We provide comprehensive insights into the quantification and distribution of Aβ deposits in the temporal Grey/White Matter, revealing a progressive increase in tandem with the severity of established diagnostic criteria (NIA-AA). We also present correlation of the Aβ load with clinical diagnosis as well as presence/absence of mixed pathology. This study introduces a reproducible workflow, showcasing the practical use of ML approaches in the field of neuropathology, with ML-based workflows offering enhanced diagnostic accuracy in routine clinical settings.

Conclusions

Acknowledging limitations, such as potential biases in the ML model, we propose avenues for future research to refine and expand the methodology. We hope to contribute to the broader landscape of neuropathology advancements, ML applications, and precision medicine, paving the way for deep phenotyping of AD brain cases and establishing a foundation for further advancements in neuropathological research.

PLATFORM 2: Tumors Glial: Part I
9
Epigenomic dysfunction and derepressed endogenous retroviral elements promote ATRX-deficient gliomagenesis

Background

Mutational inactivation of the SWI/SNF chromatin regulator ATRX occurs frequently in diffuse gliomas. Whether and how ATRX deficiency promotes oncogenesis remains unclear, despite its implication across a broad array of physiological processes. We sought to delineate the molecular mechanisms by which global epigenomic dysfunction promotes oncogenic phenotypes in ATRX-deficient glioma models.

Methods

Building on our earlier work implicating chromatin accessibility and transcriptional shifts, we employed integrated epigenomic profiling in Atrx-intact and -deficient murine neuroepithelial progenitor cells (mNPCs), validating findings will functional approaches in human glioma stem cells (GSCs) and induced pluripotent stem cell (iPSC) derivatives of appropriate genotype.

Results

We found that ATRX deficiency dramatically impacted the integrity of large heterochromatin domains genome-wide, with tangible effects on underlying gene expression. This process fundamentally altered chromatin topology, as assessed by Hi-C analysis, and associated superenhancer landscapes, influencing key developmental gene sets like the HOXA cluster. Pharmacological inhibition of HOXA signaling in GSCs selectively reduced proliferation, increased apoptosis, and impaired in vivo growth in the ATRX-deficient context. Finally, our results pointed to derepression of LINE1 endogenous retroviral elements as both key mediators and downstream effectors of ATRX-deficient heterochromatin dysfunction.

Conclusions

Our work implicates novel and targetable molecular mechanisms involving complex epigenomic rewiring and chromatin topology in ATRX-deficient oncogenesis. In doing so, we advance the understanding of a deadly brain tumor along with broader conceptions of epigenetic mechanisms as fundamental drivers in cancer.

10
Integrated clinical and molecular characterization of 200 disseminated pediatric low-grade gliomas

Background

Pediatric low-grade gliomas (PLGG) have excellent outcomes overall but are a major clinical challenge when disseminated. Diffuse leptomeningeal glioneuronal tumor (DLGNT) is a recognised entity both clinically and pathologically, however many disseminated LGG (DLGG) fall outside this diagnosis.

Methods

To better understand the clinical and molecular features of DLGG, as well as risk factors for dissemination, we assembled an international consortium of 30 sites, contributing over 200 patients with clinical annotation, along with genomic and methylation profiling.

Results

DLGG have worse progression-free (PFS) and overall survival (OS) than PLGG overall (p < 0.0001). Seventy (35%) presented with a localized mass and secondary dissemination, including some with initial gross-total resection. The most common growth pattern observed (n = 77, 40%) is a dominant suprasellar/optic pathway tumor with leptomeningeal drop-metastases involving the brainstem and spinal cord. Only 27 (14%) patients had diffuse tumors (without an identifiable dominant mass), and these had significantly worse OS (p = 0.03). The most common pathologic diagnosis was pilocytic/pilomyxoid astrocytoma (n = 92; 53%). DLGNT comprised a minority of cases (n = 23, 14%), with a strong trend (p = 0.056) towards worse survival compared to other diagnoses. The most frequent molecular alteration was BRAF fusion (78/151 with molecular testing available; 52%), followed by FGFR mutations or fusions (18/151; 12%). BRAF V600E was underrepresented (14/151; 9%). 1p deletion was rare (17 patients) but highly associated with DLGNT. Methylation classification was highly concordant with histologic diagnoses rather than clinical behavior. Importantly, patients that received upfront targeted therapies (BRAF and/or MEK inhibition, n = 9) had better PFS compared to those receiving chemotherapy (n = 146, p = 0.05). Furthermore, patients who were treated sequentially with chemo then targeted therapy had longer PFS on targeted therapy (p = 0.011).

Conclusions

This study presents the largest cohort of DLGG to date, expanding our understanding of the clinical, pathologic, and molecular features of this disease, and supports the use of upfront targeted therapy.

11
Preanalytic variables impacting clinical evaluation of PI3/AKT/mTOR signaling in diffuse glioma

Background

Biomarker-driven therapeutic trials and window of opportunity clinical trials are critical to advances in patient outcomes. Yet, evidence-based protocols that minimize preanalytic variables are critical for their successful implementation. In diffuse glioma, phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR (PI3/AKT/mTOR) signaling is an attractive therapeutic target. Yet, the relevant preanalytic variables and optimal tumor sampling methods necessary to measure pathway activity are unknown.

Methods

To investigate potential preanalytical variables we used a murine model for IDH-wildtype glioblastoma (GBM) and human tumor tissue from IDH-wildtype and IDH-mutant diffuse glioma. Cellular phosphoprotein expression was quantified from whole slide images for six phosphoproteins that are common readouts of PI3K/AK/mTOR activity (p-PRAS40, T246; p-mTOR; S2448; p-AKT, S473; p-RPS6, S240/244 and S235/236, and p-4EBP1, T37/46).

Results

First, we determined the impact of delayed time-to-formalin fixation, or cold ischemia time (CIT), on the quantitative assessment of cellular expression of six phosphoproteins that are commonly used as readouts of PI3K/AK/mTOR activity. With CITs ≥2 hours, typical of routine clinical handling, all had reduced or altered expression with p-RPS6 (S240/244) exhibiting relatively greater stability. Patient tumor samples showed a similar pattern with increased sensitivity of p-4EBP1 to delayed fixation relative to p-RPS6 (S240/244). Most clinical trials depend on the exchange of unstained slides for biomarker evaluation. Thus, we evaluated the impact of slide storage conditions on cellular expression of phosphoproteins after 3 or 5 months of storage. Slide storage at -80ºC was required to preserve p-4EBP1 and p-AKT expression and p-RPS6 (240/244) expression was not stable regardless of storage condition. Biomarker heterogeneity is an important factor impacting optimal tumor sampling. In multiple regionally distinct human tumor samples from 8 patients, quantification of p-RPS6 (240/244) expression revealed significant intratumoral heterogeneity.

Conclusions

The clinical assessment of PI3K/AKT/mTOR signaling must overcome both intratumoral heterogeneity and multiple preanalytic factors to accurately assess PI3K/AKT/mTOR signaling in diffuse glioma.

12
Activated Signaling Pathways and Therapeutic Responses of Diffuse Leptomeningeal Glioneuronal Tumor with Chromosome 1q Gain

Background

Diffuse leptomeningeal glioneuronal tumor (DLGNT) with chromosome arm 1q gain (1q+) demonstrates aggressive clinical behavior. Its rarity and the often-minute sample size limit our understanding of the biological basis of its aggressiveness. We sought to determine the activated signaling pathways and responses to current therapeutic options of 1q+ tumors in a multi-institutional cohort of 42 DLGNTs.

Methods

DNA methylation profiles of 13 1q+ tumors and seven tumors without 1q+ were compared to identify differentially methylated regions to suggest activated signaling pathways in 1q+ tumors. Orthogonal validation of the findings was performed using RNA sequencing (seven 1q+; two without), deep proteomics and phosphoproteomics (four distinct 1q+ tumors from a patient), and immunohistochemistry (18 1q+; 12 without). Treatment details and responses were available for 16 patients (nine 1q+; seven without), with progression-free and overall survival data available for all (23 1q+; 19 without).

Results

Gene Set Enrichment Analysis of all differentially methylated regions suggested increased activities of the MAPK, PI3K/AKT/mTOR, WNT, and p53 pathways in 1q+ tumors compared to tumors without. In addition, positive regulators of these pathways were hypomethylated on chromosome 1q as a putative mechanism sustaining their activation. The aberrant activation of these pathways in 1q+ DLGNT was orthogonally supported by RNA sequencing, deep proteomics and phosphoproteomics, and immunohistochemistry data. Among the overexpressed genes/proteins on 1q, CLK2 may contribute to the activated PI3K/AKT/mTOR pathway in 1q+ DLGNT. Our molecular findings suggested that 1q+ DLGNT would resist conventional chemotherapy, single-agent targeted therapy, and radiation. The cohort’s treatment response and outcome data supported this notion.

Conclusions

DLGNT with 1q+ were refractory to current treatment options. We identified the activated signaling pathways underlying the aggressiveness and treatment resistance of 1q+ tumors. Our findings suggest therapies targeting CLK2 and the MAPK, PI3K/AKT/mTOR, WNT, and p53 pathways should be further tested for 1q+ DLGNT.

13
Comparison of CDKN2A/B hom*ozygous deletion detection by DNA methylation and DNA next generation sequencing

Background

CDKN2A/B hom*ozygous deletion status is critical for grading of several central nervous system (CNS) tumors, including IDH mutant astrocytoma, pleomorphic xanthoastrocytoma, and meningioma. CDKN2A/B status can be assed using a variety of methods, but the accuracy varies due to technical and analytical variables. Here we sought to compare sensitivity of CDKN2A/B hom*ozygous deletion detection by NGS and DNA methylation array derived copy number analysis.

Methods

We retrospectively analyzed 100 CNS tumors diagnosed at NYU Langone Health between 2020 and 2023. All tumors were profiled by whole genome DNA methylation profiling using the Illumina EPIC array. Copy numbers were analyzed using the ‘conumee’ R package and visual inspection. Next-generation sequencing (NGS) analysis was performed by NYU Langone Genome PACT, a 510(k) FDA cleared (K202304) matched tumor-normal 607 gene panel. Variant allele frequency of canonical driver mutations was used to molecularly estimate tumor cell content. Discrepant cases were assed using targeted qPCR.

Results

Our cohort of 100 CNS tumors included glioblastoma (N = 75), IDH mutant astrocytoma (N = 14), oligodendroglioma (N = 4), pleomorphic xanthoastrocytoma (N = 2), meningioma (N = 1), infantile hemispheric glioma (N = 1), central neurocytoma (N = 1), diffuse midline glioma K27-altered (N = 1), and anaplastic pilocytic astrocytoma (N = 1). By DNA methylation, CDKN2A/B hom*ozygous deletion was detected in 54 cases, hemizygous deletion was detected in 13 cases, and no deletion was detected in 33 cases. By NGS, CDKN2A/B hom*ozygous deletion was detected in 31 cases, hemizygous deletion was detected in 28, and no deletion was detected in 41 cases. DNA methylation detected a hom*ozygous deletion in 23 cases that were not detected by NGS. All cases of hom*ozygous deletion detected by NGS were also detected by DNA methylation.

Conclusions

DNA methylation derived copy number assessment is more sensitive for CDKN2A/B hom*ozygous deletion than next generation sequencing. This may be due to higher sensitivity of NGS to low tumor cell content for hom*ozygous deletion calls.

14
Integrated deep learning model for prediction of DNA methylation and tumor type from histopathology in CNS tumors

Background

DNA methylation profiles, which capture the methylation status of thousands of individual CpG sites, are data-driven means to enhance diagnostic accuracy, but this technique is not yet routinely available. Deep learning of histopathology images provides an opportunity to obtain potentially useful diagnostic information quickly to assist in the subsequent workup.

Methods

Initial investigations showed that over 60,000 CpG site beta-values could be well-predicted (correlation coefficient 0.4 or higher) from histopathology images. To examine the potential of deep learning in concert with DNA methylation, we developed a model that predicts 10 major categories of CNS tumors from histopathology by integrating three distinct components: 1) prediction directly from histopathology slide images (‘direct model’); 2) predictions of DNA methylation beta-values, which are subsequently used for a classifier (‘indirect model’); and 3) routinely available patient demographics.

Results

Using a model to predict 10 categories of common CNS tumors, trained on an internal dataset of 1,796 patients, we apply this integrated model on three independent external test datasets of 2,156 patients, achieving an overall top-1 accuracy of 95% on samples which are predicted with high confidence, across the three cohorts. For individual cases, the model refined or changed the initial suspected diagnosis, which was then shown to align with the actual methylation classifier result. Additional investigations have expanded a classifier to more than 25 CNS tumor classes, with the inclusion of additional biomarker predictions (e.g., models to predict IDH, H3K27M mutation status and others) as additional features in the overall model.

Conclusions

The methylation status of a subset of CpG sites can be well-predicted from histopathology, and these inferred methylation values could then be used to classify tumors using an integrated model. Such an approach could assist pathologists in initial assessment of DNA tumor diagnosis, by providing suggestions that could then be worked up specifically for individual cases.

15
Primary diffuse leptomeningeal gliomatosis in adults: Clinical-pathological and molecular findings of 5 cases

Background

Adult primary diffuse leptomeningeal gliomatosis (PDLG) is a rare, rapidly progressive, and fatal disease marked by distinct leptomeningeal infiltration by a glial tumor in the absence of an identifiable parenchymal mass.

Methods

We describe the clinical, pathological, and molecular features of 5 adult PDLG patients (4 female, 1 male), mean age 60 years (range, 52-68).

Results

All patients had pathologic intracranial leptomeningeal enhancement at time of presentation, 3 with associated FLAIR abnormality in the adjacent brain parenchyma. Of 4 patients with available spine imaging, 3 showed spinal leptomeningeal enhancement upon presentation, and the fourth patient developed it within less than 6 months. In all patients, leptomeningeal biopsy (1 frontal, 1 temporal, 1 thoracic spine, and 2 cauda) showed infiltration by an astrocytic glioma with mitotic activity, but no definite microvascular proliferation or necrosis. GFAP and S100 were focally or diffusely positive in four cases, but negative in one case. All tumors were IDH-wildtype, 4 harboring TERT promoter mutation (1 in conjunction with NF1 mutation) and 1 with PTEN and two TP53 mutations. Chromosomal microarray showed chromosome 7 whole or partial gain in all cases, 9p loss in 4 cases (3 with hom*ozygous CDKN2A/B deletion), and chromosome 10 loss in 3 cases plus additional full and partial chromosome gains and losses, consistent with a high-grade astrocytoma (1 case) or glioblastoma, IDH-wildtype (4 cases). Methylation EPIC array was performed in 1 case and resulted in a no match. Four patients died from the disease: three while under hospice care within a month of biopsy, and one survived 1.4 years after chemoradiation therapy. Additionally, one patient remains alive with two months of post-biopsy follow-up.

Conclusions

Adult PDLG is an aggressive disease with short survival. The clinical, pathological, and molecular features closely resemble those of glioblastoma, IDH-wildtype.

16
Rapid Determination of MGMT Methylation Status in Adult-Type Diffuse Gliomas by Droplet Digital PCR Using Intraoperative Smear Preparation

Background

MGMT methylation is an important predictor of response to chemotherapy in adult-type diffuse gliomas. Currently, MGMT methylation assessment requires formalin-fixed, paraffin-embedded (FFPE) tissue. We developed a digital droplet PCR (ddPCR) MGMT assay performed on bisulfite converted DNA utilizing primers targeting CpG positions with known clinical utility, which we validated against DNA methylation array (98.7% concordance). Smear preparations are routine for intraoperative neurosurgical consultation, thus we sought to validate ddPCR using smear preparations.

Methods

Thirty-six smears were collected during intraoperative consultation. For the first 28 samples, smears were DNA extracted and bisulfite converted, and MGMT promoter methylation was quantified by ddPCR. These results were compared to DNA methylation array. For the final 8 samples, slides were H&E-stained and coverslipped for cellularity assessment prior to DNA extraction, and directly compared with corresponding FFPE specimens.

Results

Of the 28 smears, 25 (88%) were concordant with methylation of corresponding FFPE samples. Two smear preparations were falsely negative (8%). However, this was likely due to sampling as these smears did not show neoplastic cells cytologically. One smear (4%) was positive by ddPCR but negative by conventional methylation of FFPE tissue, although the ddPCR result was only marginally above threshold (4.022% vs 4%). For the eight samples with confirmed adequate cellularity, ddPCR of the smears and FFPE sections all correlated. Furthermore, the percent MGMT promoter methylation matched when normalized to tumor cellularity.

Conclusions

We describe a novel and expedited approach to MGMT methylation assessment using intraoperative smears and ddPCR in adult-type diffuse gliomas. This approach will reduce turn-around time (approximately 2 days) over conventional MGMT analysis with FFPE and could provide clinically pertinent information for treatment planning and clinical trial enrollment options prior to discharge. Furthermore, this approach may also be utilized in those cases wherein limited biopsy tissue is available, which would conserve FFPE tissue for other analyses.

PLATFORM 3: Neurodegenerative: FTLD/Lewy Body/Parkinson, Trauma, Vascular
17
ANXA11 is a significant aggregating disease protein in FTLD-TDP Type C and related TDP-43 proteinopathies

Background

TAR DNA-binding protein 43 (TDP-43) is a RNA binding protein that co-localizes to ribonucleoprotein granules tethered to lysosomes via ANXA11. TDP-43 proteinopathy occurs in 88% of amyotrophic lateral sclerosis (ALS) patients, 55% of Alzheimer’s disease patients as limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC), and 42% of frontotemporal degeneration patients as frontotemporal lobar degeneration (FTLD-TDP). FTLD-TDP pathology shows distinct histological subtypes. Type A, B and C are the most common subtypes and Type C may be the most frequent in sporadic FTLD-TDP. ANXA11 inclusions occur in ALS-associated ANXA11 variants, but ANXA11 pathology has not been described in non-variant ALS, FTLD-TDP or LATE-NC cases.

Methods

Neuropathologically diagnosed neurodegenerative disease cases (n = 818) were screened for ANXA11 variants. ANXA11 and TDP-43 immunohistochemistry and double immunofluorescence was performed on putative ANXA11 variant cases and a cohort of sporadic and genetic ALS, FTLD-TDP, LATE-NC and other neurodegenerative diseases (n = 332). Immunoblot analysis was performed on sarkosyl-insoluble fractions from post-mortem brains.

Results

TDP-43 and ANXA11 inclusions appear as thick dystrophic neurites in cortex and cytoplasmic inclusions in dentate gyrus and striatum, with similar severity in all FTLD-TDP Type C cases (n = 35). ANXA11 inclusions are uncommon in LATE-NC (n = 8, 7%), FTLD-TDP Type A and B (n = 3, 4%), and ALS cases (n = 2, 3%). In a clinical ALS patient with the ANXA11 variant p.G38R and in sporadic ALS and LATE-NC cases, ANXA11 and TDP-43 aggregates co-localize by double immunofluorescence. ANXA11 is the primary pathology in an atypical parkinsonism patient with a novel ANXA11 variant p.P75S in the absence of TDP-43, tau and alpha-synuclein pathology. By immunoblot, FTLD-TDP type C and ANXA11 variant cases show insoluble ANXA11 accumulations including a truncated fragment.

Conclusions

The identification of ANXA11 inclusions in FTLD-TDP Type C cases and related TDP-43 proteinopathies suggests that ANXA11 dysfunction is pathogenic in both sporadic TDP-43 proteinopathies and “annexinopathies” with ANXA11 variants.

18
Cellular Dissection of Multiple System Atrophy

Background

Multiple system atrophy (MSA) is a rapidly progressive neurodegenerative disease that can present clinically with atypical parkinsonism (MSA-P) or cerebellar ataxia (MSA-C). MSA has no established genetic cause and no effective treatment. Pathologically, MSA is characterized by the accumulation of alpha-synuclein in glial cells, predominantly oligodendroglia, and occasionally in subtypes of neurons. It remains unknown how populations of neurons and glia are altered by alpha-synuclein in affected brain regions in MSA patients.

Methods

We used high-throughput single-nucleus RNA-sequencing (snRNA-seq) to generate comprehensive transcriptional profiles of the cerebellum, which contains a high burden of alpha-synuclein pathology in MSA, from a large cohort of cases and age-matched controls.

Results

From the transcriptomes of over 400,000 high-quality nuclei in total, our analysis revealed statistically significant decreases in the relative abundance of specific neuronal populations, including Purkinje neurons in MSA-C. Furthermore, our preliminary results demonstrate extensive transcriptional perturbations in oligodendrocytes, including some shared cellular pathways with excitatory neurons.

Conclusions

Our approach provides a framework for understanding disease mechanisms of MSA in further studies.

19
CSF Alpha Synuclein Seed Amplification Assay Performance in Relation to Neuropathological Staging of Lewy Body Diseases and Co-pathologies

Background

AIMS To examine the predictive value of CSF alpha- synuclein seed amplification assay in an autopsy-confirmed cohort of Lewy body dementia cases

Methods

The US Dementia with Lewy Bodies Consortium (DLBC) is a NIH funded project to longitudinally assess a clinical cohort of Lewy body dementia. DLBC participants were asked to contribute CSF and blood for biomarker testing and collection for the Parkinson’s Disease Biomarker Program (PDBP) biofluid repository (BioSend). DLBC participants who died and consented for autopsy with cerebrospinal fluid (CSF) alpha-synuclein Seed Amplification Assay (SAA) results were assessed for neuropathology of Lewy Body Disease (LBD), Alzheimer’s Disease Neuropathologic Change (ADNC), Limbic Associated TDP-43 Encephalopathy (LATE) and vascular pathology.

Results

Currently, the DLBC has enrolled over 160 participants, of which 40 cases have come to autopsy. Of these, 32 are currently available for neuropathologic analysis of staging of LBD, ADNC, LATE and vascular pathology. Three cases did not have Lewy body pathology by H&E and alpha-synuclein immunohistochemistry (LB509) in brainstem, limbic or neocortical regions, and all of three were CSF alpha-synuclein SAA negative. Of the remaining 29 cases, one was SAA negative (amygdala predominant LBD), one was indeterminate (brainstem-predominant LBD) and one additional amygdala predominant LBD case was SAA positive. The remaining 26 limbic and neocortical LBD staged cases were all SAA positive.

Conclusions

CSF alpha-synuclein SAA positivity was highly predictive of limbic or neocortical stage LBD, as confirmed by neuropathologic assessment. In cases with more restricted LBD (e.g., amygdala or brainstem predominant), the SAA findings were inconsistent. Future efforts to increase the sensitivity of SAA may assist in identifying LBD in individuals in this latter circ*mstance. Importantly, the DLBC autopsy data suggest that other neurodegenerative co-pathologies do not appear to impact the ability of CSF alpha-synuclein SAA to detect limbic or neocortical LBD.

20
Neuropathology Indicates Limitations to Clinical Biomarker Based Staging of Lewy Body Disorders

Background

In recent years, proposals have been advanced to redefine or reclassify Lewy body disorders (LBDs) using clinical criteria including biomarkers. These have either minimized the differences between, or even merged, the long-established entities of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), and without systematically considering comorbid Alzheimer’s disease (AD).

Methods

We used a large dataset from the Arizona Study of Aging and Neurodegenerative Disorders to determine whether the groups defined by classical clinicopathological criteria are statistically separable. Clinicopathologically-defined groups included PD (n = 169), PD/AD (n = 83), DLB Consortium High, DLB-H (n = 64), DLB Consortium Intermediate, DLB-I (n = 173), Alzheimer’s disease (AD) with amygdala-predominant Lewy bodies, ADLB (n = 294) and AD with no Lewy pathology, ADNLB (n = 486).

Results

Analysis of variance or Chi-square tests showed highly significant (p < 0.0001) group differences in age of onset, disease duration, fraction with an apolipoprotein E4 genotype, sex, fraction with severe SN neuronal loss, fraction with peripheral alpha-synucleinopathy, brain alpha-synucleinopathy load, UPSIT smell test score, UPDRS motor score and MMSE score. Groups tended to cluster based on whether their predominant neuropathology was Lewy or AD related.

Conclusions

The ADLB and DLB-I groups are very similar to ADNLB, suggesting the Lewy pathology in these may be secondary to AD. The proposed clinical and biomarker based staging schemes for LBDs do not recognize this neuropathologically defined heterogeneity and fail to note the importance of comorbid AD. Biomarker-based LBD staging is critically needed but must incorporate AD biomarkers in order to approximate the clinicopathological classifications that have effectively guided research and treatment for decades.

21
Small Vessel Disease in the Brains of Young Military Service Members

Background

Small vessel disease, namely arteriolosclerosis, is an important contributor to neurologic morbidity. It is most noted in aged/aging individuals and particularly those with histories of hypertension and/or related cardiovascular risk factors. The Department of Defense/Uniformed Services University Brain Tissue Repository (DoD/USU BTR) is a brain bank dedicated to military brain health, and receives donations from many young Service Members (average age: 48 years) whose health records are accessible, and who have a variety of traumatic brain injury (TBI) exposures.

Methods

We blindly examined H&E-stained sections from frontal lobe (bilateral orbitofrontal and dorsolateral prefrontal), temporal lobe, basal ganglia, brainstem, and cerebellum of 50 consecutive brain donations from individuals aged ≤55 years at the DoD/USU BTR for arteriolosclerosis and perivascular hemosiderin, the severity of which was graded (0-3) on each slide.

Results

Average age was 40.9 years (range: 21-55). 29/50 cases had history of hypertension and/or related cardiovascular risk factors, e.g., diabetes, obstructive sleep apnea, and tobacco/nicotine use. 35/50 had TBI history, whether military-related (e.g., blasts) or civilian (e.g., contact sports). 39/50 had some level of arteriolosclerosis, and 49/50 had some degree of perivascular hemosiderin deposition. Of the 21 cases without hypertension or related conditions, 16 had identifiable arteriolosclerosis, 20 had perivascular hemosiderin deposits, and 15 had TBI history.

Conclusions

We present a high frequency of small vessel disease in brains of young Service Members, many of whom do not have a history of hypertension or related conditions, and many of which have TBI histories. The DoD/USU BTR represents an important opportunity to study the pathophysiology of cerebral microvascular disease over the course of aging, and to assess the contribution of various factors, namely TBI. Disclaimer: The information/content and/or conclusions do not necessarily represent the position/policy of, nor should any endorsem*nt be inferred on the part of, USU, the DoD, or the US Government.

22
Differential detection of TDP-43 in FTLD-TDP Type C using antibodies targeting distinct TDP-43 phosphorylated sites

Background

Frontotemporal dementias are neuropathologically characterized by frontotemporal lobar degeneration (FTLD). Intraneuronal inclusions of TDP-43 are the pathological hallmark of approximately half of the FTLD cases (FTLD-TDP). Based on pathologic phenotypes, FTLD-TDP cases are separated into five subtypes (Types A-E). However, the molecular factors underlying these phenotypes are still poorly understood.

Methods

Proteomic studies have highlighted the significance of TDP-43 phosphorylation in the pathogenesis of TDP-43 proteinopathies. Drawing from this premise, polyclonal antibodies (Abs) targeting TDP-43 peptide sequences that contain phospho-serines (pS) 305, 369, or 375 were developed. The objective was to ascertain if these Abs can differentially recognize TDP-43 in the frontal cortex of individuals affected by various types of FTLD-TDP (Types A-C). Immunohistochemical examinations were carried out on fixed tissue; western blot analyses, and immunoelectron microscopy were carried out on tissue-extracted sarkosyl-insoluble fractions.

Results

Initial investigations focused on FTLD-TDP Type C. Immunohistochemical studies revealed that although all Abs pS305, pS369, pS375, and pS409/410 labeled the neuropil threads, pS305 and pS369 immunolabeled a significantly higher number of them as compared to the other two Abs. Biochemical analyses revealed similar TDP-43 electrophoretic profiles using the pS305, pS369, and pS409/410 Abs, but not with the pS375 Ab. Immunoelectron microscopy studies showed the presence of TDP-43 filaments, which were recognized by the four Abs.

Conclusions

Our studies support developing and validating new Abs for TDP-43 phosphorylated sites to characterize TDP-43 in different TDP-43 proteinopathies. The current findings indicate that in FTLD-TDP Type C, TDP-43 is abnormally phosphorylated at S305, S369, S375, and S409/410. Furthermore, the observation that pS305 and pS369 Abs label a higher number of neuropil threads in Type C than pS375 and pS409/410 Abs raises questions about how these Abs label TDP-43 inclusions in the FTLD subtypes. These findings underscore the importance of extending these investigations to additional phosphorylated epitopes.

23
An analysis of cervical dorsal root ganglia hemorrhage in infant decedents

Background

Cervical dorsal root ganglia (DRG) hemorrhage is reported as a specific autopsy finding in infants with hyperextension/hyperflexion injuries and thought to occur in some cases of abusive head trauma (AHT). However, the relative sensitivity/specificity of cervical DRG hemorrhage has not been examined in larger independent cohorts and evidence of spinal nerve/axonal injury has not been reported.

Methods

A retrospective review of infant deaths examined at the Oklahoma OCME from 2015-2021 was performed, including detailed review of circ*mstances of death, cause/manner of death, and autopsy findings. A prospective analysis was also performed on 54 infant deaths, with complete neuropathology review, including cervical DRG. Axonal injury was assessed using neurofilament and beta-amyloid precursor protein (bAPP) immunohistochemistry. Iron stains were performed to assess hemorrhage organization.

Results

Cervical DRGs were examined in 20 cases of the retrospective cohort. Cervical DRG hemorrhage was present in 14/16 (87.5%) cases suspicious for AHT, and 0/4 (0%) cases without suspicion for AHT. Within prospective cohort, cervical DRG hemorrhage was present in 5/6 (83.3%) cases with suspected AHT, and only 2/48 (4.2%) cases without AHT suspicion. Axonal bAPP accumulation was observed in spinal nerve roots only in the 5 cases of suspected AHT with cervical DRG hemorrhage, but not in any other case and not in other brain regions in any case. Finally, there was discordance of hemorrhage organization between subdural and DRG hemorrhages in a subset of cases.

Conclusions

Cervical DRG hemorrhages were relatively, though not absolutely, sensitive and specific for AHT, especially when combined with the other elements of the AHT triad. There was abnormal spinal nerve bAPP accumulation in cases with suspected AHT, suggesting functional injury of cervical nerve roots that could be either mechanical and/or ischemic in nature. Organization of subdural and DRG was occasionally discrepant, suggesting a difference in the time frame of when injuries occur.

24
Diverse genomic alterations in single neurons after chronic brain trauma

Background

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with repetitive head impact (RHI) although little is known about its molecular pathogenesis. Previous studies of single neurons showed that private somatic mutations increase both during normal aging and in neurodegenerative disorders, and show diverse mutational patterns.

Methods

We applied two orthogonal single-nucleus whole-genome sequencing (snWGS) methods to neurons isolated from the prefrontal cortex of 15 individuals with CTE, and 4 individuals with RHI exposure but no CTE diagnosis, and compared mutational rates and spectra with neurons from neurotypical controls and Alzheimer’s disease (AD).

Results

We found a modest but significant elevation of somatic double-stranded single-nucleotide variants (SNVs) that resembles a pattern previously reported in AD. In addition, we found a strikingly large burden of small insertions and deletions (indels) and used duplex sequencing to show that these indels are mainly single-stranded, and again found a similar phenomenon in neurons from AD brain.

Conclusions

Our results suggest that neurons in CTE brain are exposed to stereotyped mutational processes, and that these processes are shared between AD and CTE suggesting potentially shared pathogenic mechanisms. Furthermore, the absence of similar changes in RHI neurons without CTE suggests that the development of CTE entails a mechanism beyond that caused by RHI alone.

PLATFORM 4: Tumors Glial: Part II
25
Spatial heterogeneity of glioblastoma based on methylation profiling and impact on imaging

Background

Recent advances in genome-wide DNA methylation profiling complement central nervous system tumor classification. Glioblastoma, IDH-wildtype (GBM) is characterized by molecular heterogeneity; however, intra-tumoral heterogeneity of methylation profiling is not well established. This study aimed to 1) investigate the intra-tumor spatial heterogeneity of DNA methylation subclasses, 2) investigate the spatial heterogeneity of tumor composition, and 3) examine how these affect radiologic imaging features.

Methods

We conducted methylation profiling on the EPIC array on 112 regions from 30 GBM patients (2-4 regions per patient). We applied two different methylation-based deconvolution methods, which infer the abundance of cell types in the microenvironment and malignant cell states. We derived 6084 radiomic features from multi-parametric MRI in each patient, including histograms, morphologic, and textural descriptors.

Results

Eight patients (27%) showed heterogeneity in GBM subclass on the DKFZ/Heidelberg CNS tumor classifier v12.6. The first deconvolution method showed that the dominant cell subpopulations in each region of the patient sample correspond to the DKFZ/Heidelberg-predicted classes, notably with the MES class displaying a significant myeloid cell presence. The second deconvolution method confirmed the association between the MES class and immune cell infiltration. In addition, it demonstrated an association between the two other classes and the dominant transcriptional subpopulation, with RTK I class associated with a dominant stem-like subpopulation and RTK II associated with a differentiated dominant subpopulation. Intriguingly, stem-like to differentiated cell ratio was preserved across patients, while the estimation of immune cell infiltration varied substantially across regions. We found that tumors enriched in stem-like cells exhibited distinct patterns of imaging features compared to tumors with a low abundance of stem-like cells.

Conclusions

Our findings highlight the importance of considering the heterogeneity in methylation profiling and microenvironment for developing targeted therapies and the potential of specific radiomic features for non-invasively predicting cell states and patient outcomes.

26
HOXD12 expression is linked with tumor cell stemness and a clinically aggressive subset of oligodendroglioma, IDH-mutant and 1p19q-codeleted

Background

Oligodendroglioma, IDH-mutant and 1p/19q-codeleted has the most prolonged expected survival among adult-type diffuse gliomas. However, older age is a significant risk factor. In this study, we sought to discover and characterize molecular factors that are associated with, but independently prognostic of age in oligodendroglioma.

Methods

We analyzed clinical data, DNA methylation array data, bulk and single-nucleus RNA-seq data, and single-nucleus ATAC-seq data from multiple independent oligodendroglioma cohorts, including TCGA, Chinese Glioma Genome Atlas, and cohorts published by Jonsson et al., Capper et al., Wang et al., and Blanco-Carmona et al. Patient age distributions were tested for normality. Unbiased differential gene expression, differential DNA methylation, and patient survival were analyzed in the context of patient age. Univariate and multivariate survival analyses were performed. Single-nucleus RNA and ATAC sequencing were used to analyze gene activity between and within neoplastic and non-neoplastic cells.

Results

Age at diagnosis was not Gaussian distributed in many of the datasets we interrogated. Differential gene expression analysis and subsequent DNA methylation analyses identified elevated HOXD12 gene expression and DNA gene body hypermethylation as age and survival-associated markers in multiple cohorts. Furthermore, in multivariate survival analyses, HOXD12 hypermethylation was independently prognostic of HOXD12 overexpression, patient age, radiographic features, and histologic features such as CNS WHO grade. Single nucleus transcriptomic and chromatin analyses revealed HOXD12 activity was disproportionally higher in neoplastic cells compared to microenvironmental cells, and nuclei with detected HOXD12 activity had enriched stemness scores.

Conclusions

We observed non-Gaussian age distributions in multiple oligodendroglioma cohorts, suggesting oligodendroglioma may be composed of age-associated subclasses. HOXD12 overexpression and DNA hypermethylation were associated with older patients; however, each factor was an independent risk indicator. Lastly, HOXD12 activity was highest in neoplastic cells, and its expression was associated with increased stemness, implicating a developmental biology pathway underlying its aggressive clinical phenotype.

27
2D and 3D multiplexed subcellular profiling of nuclear atypia and rupture in human glioblastoma

Background

Nuclear atypia, including altered nuclear morphology and formation of aberrant structures such as micronuclei (MN), is ubiquitous in cancer cells. Atypical primary and micronuclear envelopes can rupture leading to DNA damage, chromosomal rearrangements, and innate immune signaling. However, the causes and consequences of nuclear atypia and rupture are unknown in most cancers, including glioblastoma.

Methods

The DNA-binding protein BAF rapidly binds cytoplasmic DNA, enabling robust detection of nuclear envelope (NE) rupture. We use BAF immunohistochemistry to characterize NE rupture across major human cancer subtypes (N = 145 tumors). We perform live-cell imaging of BAF-GFP glioblastoma patient-derived cell lines (PDCL) (N = 4) to analyze the spatiotemporal dynamics of NE rupture. Using highly-multiplexed 2D cyclic-immunofluorescence (CyCIF), we analyze the causes, consequences, and spatial organization of NE rupture in glioblastoma tissue microarrays (N = 145) and autopsies (N = 4). We develop 3D-super-resolution multiplexed immunofluorescence methods to analyze nuclear morphology and correlate with NE rupture, DNA damage, and immune signaling in GBM tissues (N = 5).

Results

BAF immunohistochemistry demonstrates NE rupture in all cancer subtypes, with particularly high rates in glioblastoma (up to > 50% of cells). Live-cell imaging of PDCL demonstrates frequent rupture and repair of the NE. 2D and 3D multiplexed imaging of GBM tissues shows that primary and MN rupture are associated with irregular nuclear morphology, low lamin A/C and B1 expression, DNA damage, and activated cGAS-STING and interferon signaling. Multiplexed imaging also reveals lower lamin levels and increased NE rupture in neural-progenitor-cell-like (NPC-like) cells relative to other lineages. Spatial analysis shows that NPC-like cells are predominantly distributed to tumor infiltrating edges.

Conclusions

Nuclear atypia and rupture are core features of human cancer. In glioblastoma, NE rupture is associated with irregular nuclear morphology, reduced lamin expression, NPC-like differentiation, DNA damage, and activation of cGAS-STING/interferon signaling. NE instability and rupture likely contribute to tumor progression and may represent an intrinsic vulnerability in glioblastoma.

28
H3K27M mutation loss in a recurrent diffuse midline glioma

Background

We present the case of a 32-year-old male with a history of a thalamic diffuse midline glioma, H3K27-altered (confirmed with molecular analysis). Subsequent recurrence in the right frontal lobe was notable for the absence of H3K27M mutation (confirmed with molecular analysis).

Methods

Immunohistochemical staining and subsequent molecular analysis were performed on the initial thalamic resection specimen and the subsequent frontal lobe resection specimen.

Results

The initial thalamic specimen was found to have a histone H3 mutation (H3K27M) and a FGFR1 mutation (N577K). Status post radiation and chemotherapy, the patient was found to have a T2/FLAIR hyperintense enhancing focus in the right frontal lobe with interval increase in enhancement. Following resection, the recurrent tumor exhibited morphology similar to the initial resection. However, the immunohistochemical profile of the recurrent neoplasm was unexpected; there was variably retained H3K27 trimethylation and negative H3K27M staining. Molecular analysis failed to detect an H3K27M mutation, but the FGFR1 mutation (N577K) was present. The patient went on to develop additional frontal lobe lesions. Despite extensive disease, the patient survived for two years after initial diagnosis.

Conclusions

Notably, the CNS WHO 2021 states that “[s]omatic heterozygous H3 p.K28 (K27) mutations are invariably maintained during the disease”. The current case demonstrates that the H3K27M somatic mutation is not invariably maintained in recurrence and raises the possibility that a sub-clonal population of tumor cells exist in diffuse midline gliomas that lack the canonical molecular profile of a diffuse midline glioma.

29
Histopathological and Molecular Heterogeneity of Tumors classifying as Dysembryoplastic Neuroepithelial Tumour (DNT) by DNA Methylation

Background

Dysembryoplastic neuroepithelial tumor (DNT) is a glioneuronal neoplasm in the cerebral cortex that occurs in children or young adults and is characterized by activating mutations of FGFR1. The differential diagnosis of DNT is wide, including neoplastic to non-neoplastic. DNA methylation can be used for diagnosis. We aimed to analyze the heterogeneity of lesions classifying as DNT by DNA methylation.

Methods

We analyzed clinical, pathological, and molecular data of 46 tumors that classified as DNT methylation class using the Heidelberg DNA methylation classifier. Results of mutational, copy number and gene fusion analyses were retrieved from clinical next-generation sequencing reports including Oncomine (DNA&RNA), NYU Genome PACT (DNA) and NYU FusionSEQer (RNA). DNA methylation calibrated score was correlated with sample quality, clinical, pathological, and molecular results.

Results

Tumors classifying as DNTs with methylation scores >0.9 (23 cases) carried histological diagnoses such as diffuse astrocytoma, oligodendroglioma, and papillary glioneuronal tumor and showed a flat profile in copy number (18 cases, 78%). DNTs with scores < 0.9 (23 cases) had a variety of histological diagnoses ranging from supratentorial ependymoma to focal cortical dysplasia. Other than FGFR1-TACC1, we detected GPRC5B-PRKCA, TNS3-ETV1, and GNAI1-BRAF fusions. Furthermore, low score tumors had a higher number of CN variations (4 cases with >3 gene alterations), most frequently gain of PDGFRA. Overall, 16/46 (35%) of the histological diagnoses were correctly reclassified as DNTs by DNA methylation.

Conclusions

DNA methylation can accurately classify tumors that are histological mimickers of DNT. However, our data suggest that brain tumor lesions classifying as DNT by DNA methylation with low score represent a diverse group. Furthermore, some FCD can classify as DNT, albeit with low calibrated score and require close correlation.

30
Hypoxia and macrophage-secreted TGF-β1 support glioma stem cell enrichment in the peri-necrotic zone of glioblastoma

Background

After necrosis develops in glioblastoma, IDH-wildtype (GBM), the tumor microenvironment undergoes significant remodeling. The peri-necrotic zone (PNZ) is characterized by severe hypoxia, influx of tumor-associated macrophages (TAMs), and enrichment of glioma stem cells (GSCs). In prior bioinformatic investigations, Hippo transcriptional co-activators YAP/TAZ have been implicated as master regulators of stemness in the PNZ. We propose that GSCs are enriched through the activation of YAP/TAZ, both directly by hypoxic regulation and by TAM secretion of TGF-β1.

Methods

ELISA of primary macrophage conditioned media and in-silico analyses of GBM and macrophage single-cell RNAseq (scRNAseq) data using NichenetR were performed to determine regulatory cytokines specific to hypoxia. Primary GBM neurospheres were exposed to normoxia, hypoxia, and cytokine candidates, and whole cell, cytoplasmic, and nuclear lysates were analyzed for YAP/TAZ, Hippo readouts (AXL, CYR61), and targets that may regulate stemness. Extreme limiting dilution assays (ELDA) assessed GSC stemness.

Results

Under hypoxia, YAP/TAZ translocated to the nucleus while Hippo readouts AXL and CYR61 were upregulated. Hypoxia likewise upregulated Inhibitor of Differentiation 2 (ID2), a YAP/TAZ target that potentially regulates GSC stemness. Conditioned media from hypoxic primary macrophages also caused upregulation of Hippo readouts, and cytokine analyses showed secretion of IL-8, CXCL5, EGF, and TGF-β1. Computational analyses using NichenetR predicted that hypoxic macrophage-secreted TGF-β1 had the highest regulatory potential among ligands that interact with hypoxic tumor cells and was strongly associated with Hippo target upregulation. Dose-response analysis of TGF-β1 on GBM showed stepwise upregulation of YAP/TAZ translocation and CYR61 under normoxia; GBM treated with TGF-β1 in hypoxia show an additive effect. Using ELDA, we found that TGF-β1 alone enhances GSC stemness and also synergizes with hypoxia.

Conclusions

Hypoxia and TGF-β1 activate Hippo transcriptional activity and enhance maintenance of GSCs, likely contributing to GBM progression and therapy resistance.

31
PDE10A haploinsufficiency activates PI3K/AKT signaling independent of PTEN to induce an aggressive glioma phenotype

Background

Glioblastoma is characterized by frequent somatic copy number alterations (SCNAs) harboring oncogenes and tumor suppressors. In this study, we employed a multi-faceted approach, integrating computational analyses and experimental validations, to uncover a novel clinically relevant tumor suppressor in glioblastoma.

Methods

Logrank survival analysis for SCNAs was performed in the TCGA glioblastoma dataset. Findings were validated in two additional datasets (NYU, REMBRANDT). Combining data from a CRISPR/Cas9 functional screen (neural stem cells/glioma stem cells) with transcriptional data from normal brain (GTEx) and glioblastoma (TCGA, CGGA, IvyGAP), we nominated a potential haploinsufficient tumor suppressor gene in the SCNA region of interest. Utilizing the RCAS/tv-a mouse model of glioblastoma, we tested the nominated gene for its effects in vivo (survival, grade, proliferation) and in vitro (neurosphere formation, proliferation, treatment response). We then combined single nucleus sequencing of mouse gliomas with in vitro assays (Western blot, cell adhesion/migration, pharmacologic pathway inhibition) to determine mechanism of action.

Results

We discovered cytoband 6q27 as an independent poor prognostic marker across multiple glioblastoma datasets. Combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data nominated phosphodiesterase 10A (PDE10A) as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased Pi3k/Akt signaling in a Pten-independent manner. Single nucleus RNA sequencing from mouse gliomas, in combination with cell culture validation further showed that Pde10a suppression was associated with a proneural to a mesenchymal transition that exhibited increased cell adhesion and decreased cell migration.

Conclusions

Our results emphasize the clinical implications of PDE10A loss, identifying a subset (approximately 32%) of glioblastoma patients with worse outcomes, heightened resistance to standard-of-care therapy, and potential sensitivity to PI3K inhibition.

32
Prognostic impact of CDKN2A deletion in oligodendroglioma

Background

Oligodendrogliomas represent approximately 5-10% of gliomas. While molecular advances have defined this entity as 1p/19q-codeleted and IDH-mutant, further studies aimed at molecular markers for grading have had conflicting results. Additionally, studies prior to 2016 often included gliomas mimicking oligodendrogliomas, confounding interpretation of their results.

Methods

We reviewed a series of 184 oligodendroglioma specimens taken at initial resection or recurrence All specimens evaluated were grade 2 by clinical grading criteria at time of diagnosis. Whole exome sequencing (WES) was performed on all specimens, allowing confirmation of 1p/19q-codeletion by inferred copy number variation, as well as IDH-mutant status. Copy number for CDKN2A was inferred from WES data and, in those with potential hom*ozygous loss, CDKN2A status was confirmed by fluorescence in situ hybridization (FISH).

Results

Two of the 107 grade 2 oligodendrogliomas that were evaluated at initial diagnosis had hom*ozygous CDKN2A loss (2%). The median overall survival from time of diagnosis for patients who tumors demonstrated hom*ozygous CDKN2A loss was 46 months while those with intact or hemizygous CDKN2A status had a median overall survival of 252 months. Five of the 77 oligodendrogliomas evaluated at tumor recurrence had hom*ozygous loss of CDKN2A (6%). In this population, the median overall survival from time of initial diagnosis for those whose tumors demonstrated hom*ozygous CDKN2A loss at recurrence was 80 months while those with intact or hemizygous CDKN2A status had a median overall survival of 305 months. Further studies are ongoing, including expanding the cohort by an additional 50 specimens to increase the power, as well as additional analysis of WES data to identify mutations in genes of interest, and any relationship to overall survival. Covariate analysis is also ongoing.

Conclusions

Our current univariate analysis provide support for using CDKN2A status as a grading criteria for oligodendroglioma.

PLATFORM 5: Neurodegenerative: Other
33
Multi-OMIC analysis of Huntington disease reveals a neuroprotective astrocyte state

Background

Huntington’s disease (HD) is an incurable neurodegenerative disorder caused by an expansion of CAG repeats in exon 1 of the HTT gene. The disease features neuronal loss and astrogliosis most severely involving the striatum, with spiny projection neurons (SPNs) in the caudate and putamen being severely depleted. Nonetheless, neurodegeneration in HD extends to other brain regions including the cerebral cortex, where specific populations of excitatory neurons are selectively lost. To date, the underlying mechanisms of HD’s selective regional vulnerability remains to be fully understood. In this work, the roles of astrocytes in mediating selective regional vulnerability to neurodegeneration will be explored.

Methods

We used a combination of bulk RNAseq, lipidomics, and single nucleus RNAseq on 76 samples from 11 control brains and 20 HD brains. We also performed validation studies using multiplex immunohistochemistry, and in vitro cultures including murine and patient derived neurons.

Results

Using a multi-omic approach, we identified a gene signature driven by the HTT gene CAG repeat length, enriched in astrocytic genes, which implicated fatty acid metabolism. Lipidomic analysis highlighted correlations between lipid species and HD grade, and integration of bulk RNAseq and lipidomics implicated pathways related to protein folding and cell death. Single nucleus RNAseq analysis of over 40,000 astrocytes identified two astrocyte types: protoplasmic and fibrous-like CD44-positive astrocytes. In HD, metallothionein-enriched protoplasmic astrocytes in the caudate were depleted. The abundance of the metallothionein-rich astrocyte cluster was correlated with the abundance of vulnerable SPNs. Conversely, metallothioneins were elevated in HD cingulate astrocytes. Genome wide association studies showed a correlation between elevated metallothionein levels and delayed age of onset in HD. Functional studies demonstrated neuroprotective effects of metallothionein-overexpressing astrocytes in co-culture models with patient derived HD medium spiny neurons.

Conclusions

Our findings unveil a regionally-enriched astrocytic phenotype in less vulnerable brain regions that could be harnessed for neuroprotection in HD.

34
Chronic Traumatic Encephalopathy in a Cohort of Military Veterans in the UNITE Brain Bank

Background

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disease associated with exposure to repetitive head impacts occurring in contact sport and military-related activities. CTE has also been described after blast exposure.

Methods

Here, we report the demographic, clinical, and neuropathological characteristics of a case series of military veterans whose brains were donated to the UNITE Brain Bank. Informants for donors participated in a battery of online questionnaires and phone interviews to retrospectively assess clinical information and athletic and military histories. Neuropathological diagnoses of CTE and other neurodegenerative diseases were based on well-established criteria. Clinical and neuropathological evaluations were conducted blinded to one another.

Results

Among 307 brain donors, 302 (98.4%) of whom were male, mean (SD, interquartile range) age at death was 71.45 (17.43, 65-84) years. Most donors (262 [85.3%]) played contact sports, including 234 (76.2%) who played American football. 84 (27.4%) donors saw combat, 80 (26.1%) suffered military-related traumatic brain injuries (TBI) and 45 (14.7%) were exposed to blasts. 179 (58.3%) donors were diagnosed with CTE. Alzheimer’s disease was diagnosed in 84 (27.4%), Lewy body dementia in 68 (22.1%), frontotemporal lobar degeneration in 21 (6.8%), and motor neuron disease in 1 (0.3%). Among 179 with CTE, 175 (98%) were also contact sport athletes. Among the 4 (2.3%) donors with CTE who did not play contact sports, 4 (100%) saw combat, 4 (100%) suffered military-related TBI and 3 (75%) were exposed to blasts. Informant-reported post-traumatic stress disorder symptoms were present in 120 (39.1%) donors, with a similar frequency (42.5%) among those with CTE. Dementia was present in 203 (66.1%) and was more common (74.3%) among those with CTE.

Conclusions

Military brain donors with a history of contact sport play had a high rate of CTE. Contact sports and military service are independent risk factors for CTE, future studies will explore their potential cumulative effect.

35
Deciphering the role of the NBIA-mutated protein WDR45 in autophagy and neuroprotection

Background

Perturbations in autophagy have been implicated in various neurodegenerative disorders including Parkinson’s and Alzheimer’s disease. WDR45, an autophagy gene is mutated in a group of patients with the familial neurodegenerative disease – Neurodegeneration with Brain Iron Accumulation (NBIA). This disease is characterized by neuronal loss and iron accumulation in basal ganglia and other parts of brain. WDR45 belongs to the WIPI family of proteins with four mammalian members. The precise biological role of WDR45 and how its disruption causes neurodegeneration is unclear.

Methods

To shed light on the function of WDR45, we have generated a deletion mutant for the Drosophila melanogaster WDR45 hom*olog – CG11975.

Results

These mutants show markedly diminished life span and compromised flight ability. There is extensive accumulation of Ref2 and ubiquitin-positive aggregates in the brains of these animals. These findings suggest that the fly WDR45 mutants may serve as a good model to understand the molecular mechanisms of NBIA. Using a proteomics approach, we have identified putative autophagy cargos that may be degraded with the help of WDR45.

Conclusions

By examining the functional interaction between WDR45 and its putative cargos, we hope to provide novel insights into the physiological roles of autophagy in the brain and the significance of these processes for neurodegenerative diseases, particularly NBIA.

36
The New Brunswick Neurological Syndrome of Unknown Cause (NSUC) -- Evidence of a Fake

Background

On March 17, 2021 a press conference in Canada aired featuring a Moncton neurologist and New Brunswick’s Chief Medical Health Officer, regarding a cluster of 48 patients in New Brunswick, who had symptoms reminiscent of CJD, and were claimed to have a new neurological disease. Their onset of disease was between 2015 and 2021. 46 of those patients had at that time been reported by aforementioned neurologist. Further news publications suggested that environmental factors were causing this disease: initially a zoonosis of Chronic Wasting disease, and subsequently as beta-methyl-amino-L-alanine from algae bloom. This news has significantly disturbed the (inter)national medical community and the population of New Brunswick.

Methods

Between 2019 and 2021 10 patients died that belonged to the cluster. 8 patients underwent autopsy. 18 brain tissue blocks were examined using H&E, Luxol-PAS, B-Amyloid, Tau, P62, TDP43, Synuclein, and PrP. Frozen tissue of 7 cases processed for PrP Western blot.

Results

The neuropathological exams of 8 showed one case of metastatic carcinoma, one with FTLD-TDP43, two with neocortical Lewy body pathology, 2 cases of Alzheimer’s disease, one case of mainly vascular dementia, and one case without significant pathology (consistent with patient’s medical history). In these 8 patients no evidence for a prion disease was found by IHC or Western blotting, nor novel pathology.

Conclusions

Statistical evaluation revealed that the varied diagnoses present, predicted similar variability in the entire cluster of 48 patients at a p = 0.0001 level (using classical probability theorem methodology). On June 3, 2021 the Oversight Committee was established in New Brunswick reviewing the clinical and epidemiological data of the 48 patients. They ultimately concluded in 2022 that NSUC did not exist. Unfortunately, the aforementioned neurologist still professes the existence of this ‘new disease’, now with as cause herbicide toxicity, and is still supported by news outlets and reported internationally.

37
Efficacy of Routine Scout Sections in the Diagnosis of Spongiform Encephalopathies

Background

Prionopathies are rare CNS diseases involving aggregation of misfolded prion protein (PrP). They include sporadic/iatrogenic/familial Creutzfeldt-Jakob disease (CJD), variant CJD, other genetic forms like Gerstmann-Straussler-Scheinker disease (GSS), and more. Due to anticipated risk of infectivity, advanced precautions are undertaken in centres handling suspected cases, with some taking limited sampling (scout sections) to render a preliminary diagnosis before referring the brain to a subspecialized centre with dedicated equipment and resources. In Canada, this is the CJD Surveillance System (CJDSS), which receives all such cases nationwide.

Methods

Even sampling scout sections (frontal cortex, temporal cortex, and cerebellar vermis) demands intensive resource commitment. Therefore, it is of interest to compare the accuracy of evaluating scout sections against the diagnosis rendered by the CJDSS. In the London Health Sciences Centre database, there were 111 completed referrals: 88 were positive for a prionopathy (86 sporadic/iatrogenic/familial CJD, 1 GSS, and 1 sporadic panencephalic CJD) and 23 had no evidence of a prionopathy (all of which were correctly determined as such).

Results

Of the 88 confirmed cases, 6 were suspicious for CJD with notable uncertainty due to equivocal PrP immunohistochemistry (IHC), concomitant neurodegenerative diseases, and/or lack of histological findings with clinical suspicion of disease involvement contralateral to the side sampled. The scout sections couldn’t delineate specific rarer prionopathies (GSS, sporadic panencephalic CJD), but could distinguish them from typical cases. In some CJD cases, vacuolation was relied upon in place of unreliable PrP immunopositivity. Frontal and temporal cortices were often co-involved, with occasional differences in PrP IHC intensity. Several cases demonstrated isolated involvement of the vermis by IHC-positive plaques and unremarkable neocortices, supporting the necessity of cerebellar inclusion in routine sampling.

Conclusions

Overall, scout samples demonstrate considerable efficacy in the diagnosis and triaging of suspected prionopathy cases, though with some limitations incurred in artifactual immunostaining, multiple disease processes, and rarer prionopathies.

38
Differing cognitive profiles in limbic-predominant age-related TDP-43 encephalopathy, frontotemporal lobar dementia, and Alzheimer disease

Background

Limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC) has recently been recognized as a neuropathologic entity distinct from frontotemporal lobar dementia (FTLD). It is characterized by transactive response DNA-binding protein of 43-kDa with (TDP-43)-immunoreactive inclusions that originate in the amygdala and then progress to the hippocampusi and the middle frontal gyrus. LATE-NC may mimic Alzheimer disease clinically and often co-occurs with Alzheimer disease neuropathologic change (ADNC). We aim to focus on the cognitive effects of isolated and concomitant LATE-NC in comparison to and ADNC and FTLD-TDP.

Methods

Cognitive/neuropsychological, neuropathologic, and demographic variables were analyzed in 28 control, 31 isolated LATE-NC, 62 isolated FTLD-TDP, and 244 isolated ADNC, and 172 concurrent LATE-NC/ADNC subjects from the National Alzheimer’s Coordinating Center, as well as a cohort of 2,210 with varying mixed neuropathologies using multivariate logistic regression analysis to disentangle the relative contribution of each disease process and uncover the clinicopathologic characteristics of each neuropathologic entity.

Results

Cases with LATE-NC and ADNC were significantly older than controls, while FTLD-TDP cases were significantly younger. The presence of hippocampal sclerosis was significantly associated with LATE-NC but not ADNC or FTLD-TDP. Both LATE-NC and ADNC exhibited deleterious effects on overall cognition proportional to their neuropathological stages; concurrent LATE-NC/ADNC exhibited the worse overall cognitive effect than either process in isolation. ADNC and FTLD-TDP were associated with impairment in all cognitive domains, while LATE-NC had significant deleterious effects on logical memory and some measures of language and executive function. Multivariate logistic regression analysis determined an independent risk of cognitive impairment for LATE-NC (OR 1.71;p=0.0051), ADNC levels (OR 5.72; p < 0.0001), and FTLD-TDP (OR 14.30; p < 0.0001).

Conclusions

These data add to the existing knowledge on the clinical consequences of LATE-NC, ADNC, and FTLD-TDP pathology and the growing literature on the effects of multiple concurrent neurodegenerative pathologies.

39
Brain Digital Slide Archive (BDSA): An Open Source Whole Slide Image Sharing Platform for AD/ADRD Research and Diagnostics

Background

Neuropathologic evaluation of brain tissue is crucial for diagnosis, yet sharing such data is difficult due to inconsistencies in sample processing and digital pathology analysis across different sites. However, slide digitization and machine learning offer ways to standardize and improve diagnostic accuracy. Building upon the Digital Slide Archive – a versatile platform focused on cancer image analyses – we aim to provide a platform geared to the neurodegenerative neuropathology community, creating a federated open-source Brain Digital Slide Archive (BDSA) platform.

Methods

Our objective is to support brain whole slide images (WSIs) for AD/ADRD research compliant with FAIR (findable, accessible, interoperable, reusable) data principles. FAIR metadata for ADRC participants will be searchable through the NACC Data Front Door. We are developing workflows for sharing WSIs, annotations, and metadata, and enabling training and deployment of image analysis algorithms on multi-institutional datasets. We aim to address variability in practices through harmonized data-driven diagnostic thresholds. WSI data are sourced from an international group comprising 9 AD/ADRD-focused centers. Our goal is to integrate multiple cohorts, datasets, and model training results within a unified online portal.

Results

We convey challenges and opportunities related to harmonizing WSI data across multiple sites which is a “work in progress”. We are crafting software processes specific for AD/ADRD WSIs, performing regular software testing, and incorporating administrative best practices (e.g. universal data use agreements). This will enable the development of secure, high-quality research tools while maintaining control of WSIs at individual institutions. Ultimately, the BDSA will feature standardized and shared scientific, diagnostic, and didactic applications across sites.

Conclusions

We are creating a standardized, accessible, and searchable digital repository of neuropathological data. By providing infrastructure, standards, and reference workflows, we will promote access to, and utilization of, histologic and clinical data to advance our understanding of neurodegeneration.

40
Pathologically Based Criteria to Distinguish Essential Tremor from Controls: Analyses of the Human Cerebellum

Background

Essential tremor (ET) is among the most prevalent neurological diseases. Diagnosis is based entirely on neurological evaluation. Historically, there were few postmortem brain studies, hindering attempts to develop pathologically based criteria to distinguish ET from control brains. However, an intensive effort to bank ET brains over recent years has resulted in postmortem studies involving >200 brains, which have identified numerous degenerative changes in the ET cerebellar cortex. While ET and controls have been compared with respect to individual metrics of pathology, we now sought to derive a combination of metrics to distinguish ET from controls and determine how well this combination performs.

Methods

Analyses included 100 ET brains from the Essential Tremor Centralized Brain Repository and 50 control brains. A standard tissue block from the cerebellar cortex was used to quantify 11 metrics of pathological change that reflected changes in the Purkinje cell and related neuronal populations. Three supervised classification algorithms were investigated, including logistic regression with ridge penalty, random forest and gradient boosted decision tree. Data were divided into training and validation samples

Results

All three algorithms performed similarly to correctly predict a diagnosis of ET. The strongest pathologic predictors were those based on Purkinje cell (PC) loss, climbing fiber abnormalities, PC axonal torpedoes and PC heterotopias. Using logistic regression with a ridge penalty algorithm, the simplest method, sensitivity ranged from 87.5% to 96.64%, with six of eight values >95%, and the specificity ranged from 92.87% to 98.11%, with five of eight values >95%. We developed a web-based application that uses these metric values, and based on specified cut-offs, determines the likely diagnosis.

Conclusions

These analyses illustrate the ability to correctly predict a diagnosis of ET and set the stage for use of pathologically based criteria to distinguish clinically diagnosed ET cases from controls at the time of postmortem.

PLATFORM 6: Tumors Nonglial
41
Whole genome cytogenetic analysis and DNA panel sequencing identifies recurrent genetic alterations in epigenetic pineoblastoma subtypes

Background

Analyses of methylation profiles led to the identification of distinct epigenetic pineoblastoma (PBL) subtypes. The aim of this study was to systematically analyze the genetic alterations of these pineoblastoma subtypes.

Methods

Cytogenetic alterations of tumor samples of 76 patients with a diagnosis of PBL confirmed by reference neuropathology and methylation-based classification were analyzed by high-resolution genome-wide molecular inversion probe (MIP) analysis. 72 cases were screened for mutations by next-generation DNA panel sequencing.

Results

Referring to the proposed epigenetic PBL subtypes (Liu et al., 2021), 48 PBL-miRNA1 (1A=40; 1B=8), 17 PBL-miRNA2, 4 PBL-MYC/FOXR2, and 7 PBL-RB1 samples were identified in our cohort. PBL-miRNA subtype tumors carried characteristic alterations in microRNA-processing genes (DICER1 mutations (n = 18), hom*ozygous deletions of DROSHA (n = 17) and DROSHA mutations (n = 12). Most frequent cytogenetic aberrations in PBL-miRNA were whole chromosome (chr.) 7 gain (n = 30) and chr. 14 loss (n = 21). DICER1 mutations were significantly associated with chr. 14 loss (p < 0.001). 21 cases showed gains of chr. 14, and 8 further cases focal gains of the OTX2 oncogene (chr. 14q). 15 cases of the miRNA subtypes displayed polyploid karyotypes.

Conclusions

The epigenetically defined PBL-miRNA subtypes are characterized by distinct cytogenetic and mutational events. Frequent copy number gain of OTX2 may indicate a potential oncogenic role in the pathogenesis of PBL.

42
Molecular testing rarely changes the grade of meningiomas: The two-year experience at a single tertiary care center

Background

According to the fifth edition of the WHO classification of central nervous system tumors, meningiomas are designated as grade 3 in the presence of a TERT promoter mutation or CDKN2A/B hom*ozygous deletion regardless of histologic features. Our institution changed its practice to include molecular profiling of all meningiomas following publication of these criteria. We reviewed meningiomas at our institution that underwent molecular testing to determine the frequency with which histologically grade 1 or 2 meningiomas became molecularly re-classified as grade 3. We also reviewed cases that were molecularly upgraded to determine whether there is justification for being selective for which meningiomas undergo molecular profiling.

Methods

From January 2022 to February 2024, our institution molecularly characterized 225 consecutive meningiomas from 223 patients. TERT promoter mutational analysis was done by Sanger sequencing and CDKN2A/B status was determined by FISH or chromosomal microarray.

Results

By histology alone, 137 cases (60.9%) were grade 1, 81 (36.0%) were grade 2, 6 (2.7%) were grade 3, and one was not graded. Of the histologic grade 1 meningiomas, only one case (0.73%) showed grade 3 molecular features, with a TERT promoter mutation, 1p loss, and an intragenic DMD deletion but intact CDKN2A/B. Of the histologic grade 2 meningiomas, two cases (2.5%) showed grade 3 molecular features. One case exhibited a TERT promoter mutation, CDKN2A/B hom*ozygous deletion, and 1p loss. The other case had a TERT promoter mutation only. The histologic grade 1 meningioma that was molecularly upgraded showed worrisome radiographic features, including an irregular interface between the tumor and adjacent brain and evidence of rapid growth with vasogenic edema and midline shift, despite absence of brain invasion on reviewed slides.

Conclusions

In our series, molecular testing was most indicated in histologic grade 2 meningiomas and helpful in a histologic grade 1 meningioma with radiographic features suggestive of aggressive behavior.

43
Clinical and Molecular Landscape of TERT Promoter Mutant Meningiomas

Background

TERT promoter (TERTp) mutation in meningiomas has been associated with shorter survival and worse prognosis. The 2021 WHO CNS Classification designated TERTp mutation as a molecular biomarker of WHO Grade 3 Meningioma. However, it is unclear whether TERTp-mutant meningiomas represent a clinically and molecularly hom*ogenous group and how they interact with other molecular biomarkers.

Methods

We analyzed clinical and molecular data of 497 meningiomas from eight institutions that underwent next-generation TERTp sequencing. Copy numbers (CN) were derived from DNA methylation data using the Conumee package. Clinical outcomes were assessed from medical records.

Results

Thirty meningiomas harbored TERTp mutation, all WHO 3, and 468 were TERTp wildtype, of which 21 were WHO 3. Of the TERTp mutants, 50% were in women and 50% in men, with a median age of 66 (range 47-82) years. 76.7% (22/30) of TERTp mutant meningiomas recurred, with a median time to recurrence of 9.5 (range 0.5-175) months. The average mitotic count of TERTp mutants was 9.5 mitoses per 10 high-powered fields. CDKN2A/B locus was lost in 45.8% of TERTp mutants, with hom*ozygous loss in 37.5%, and hemizygous loss in 8.3%. Grade 3 meningiomas with or without TERTp mutations did not differ in terms of recurrence (p = 0.75) or survival (p = 0.99). TERTp-mutant meningiomas with CDKN2A/B hom*ozygous deletions demonstrated a significantly shorter post-operative time to progression compared with TERTp-mutant tumors with intact CDKN2A/B (median recurrence time 7 vs. 29 months, p < 0.02). Nearly all TERTp-mutated meningiomas (95.8%) showed concurrent CN aberrations, with frequent loss of chromosomes 1p (95.8%) and 22q (91.3%).

Conclusions

TERTp mutation rarely occurs in the absence of other molecular markers of aggressive behavior in meningiomas. The effect of TERTp mutation in WHO grade 3 meningiomas is influenced by these additional alterations, with concurrent CDKN2A/B hom*ozygous loss associated with significantly earlier progression.

44
Identification and functional phenotyping of lymphoid aggregates in brain metastases

Background

Lymphoid aggregates (LA), such as tertiary lymphoid structures (TLS), have been shown to predict overall survival and response to immune checkpoint therapy in non-CNS cancers. Data on tumors of the CNS, however, are sparse.

Methods

Here, we applied CD20 bright-field immunohistochemistry as a screening tool to identify LA in a retrospective cohort of 461 FFPE-brain metastasis samples, derived from the archives of the University Cancer Center (UCT) Frankfurt, Germany, between 2015 and 2022. Stereotaxic biopsies as well as spinal/vertebral metastases were excluded from the study. LA-positive tumors were further analyzed by multispectral whole slide TSA-immunofluorescence (IF) using antibodies for von Willebrand factor, CD3, CD20, CD163, Lamp3, pan-cytokeratins, and DAPI as counterstain. TSL-positive tumors were further characterized by whole exome sequencing, bulk RNA sequencing, spatial transcriptomics and customized highplex-sequential IF panels that included > 40 antibodies for the analysis of tumor immunology and microenvironment.

Results

TLS were observed in 38% of brain metastases and were most common in lung to brain metastasis (46%). Our preliminary data revealed a survival benefit in patients with TLS+ brain metastasis. By RNAseq, spatial transcriptomics and highplex-IF, different types of TLS were identified, based on their cellular and molecular compositions. Interestingly, most of the TLS identified lacked the structural organization reminiscent of a germinal center, suggesting an abrogated immune response.

Conclusions

Further analysis of the TLS subtypes is being conducted to investigate their biological significance and clinical relevance in patients’ response to therapy.

45
FOXR2-overexpressing CNS tumors exhibit substantial diversity across histological, molecular, and clinical dimensions

Background

FOXR2 activation is regarded as the pathognomonic molecular signature of CNS neuroblastoma. However, a comprehensive understanding of the landscape for CNS tumors exhibiting FOXR2 activation is lacking.

Methods

Histopathologic, molecular, and clinical data analysis of 36 FOXR2-overexpressing CNS tumors (expression > 1.5 times the interquartile range above the third quartile of all CNS tumors) identified through screening of institutional dataset.

Results

FOXR2-overexpressing CNS tumors showed a broad anatomic distribution throughout the neuraxis. Of the 36 tumors, 17 (47.2%) were gliomas and 17 (47.2%) were embryonal tumors. The gliomas included 16 high-grade gliomas (eight histone H3 K27M-mutant diffuse midline gliomas, seven radiation-associated tumors, and one with MN1::PATZ1 fusion) and one pilocytic astrocytoma. The embryonal tumors included ten CNS neuroblastomas, six pineoblastomas, and one medulloblastoma. Two were difficult-to-classify high-grade neuroepithelial tumors. The gliomas and embryonal tumors demonstrated comparable FOXR2 expression. FOXR2-overexpressing gliomas, but not embryonal tumors, displayed diverse concomitant genetic alterations. The most common mechanisms of FOXR2 activation involved structural alterations causing promoter donation from other genes, frequently incorporating non-canonical non-coding exons, followed by activation of exon -3 promoter or LINE-1 retrotransposon-driven expression. Tumor location, histology, and concomitant alterations correlated with patient outcomes. While FOXR2-overexpressing gliomas were aggressive (5-year overall survival 13.3%), FOXR2-overexpressing embryonal tumors showed a diverse prognosis. FOXR2-activated CNS neuroblastoma showed a 5-year overall survival of 100%. Pineoblastomas with FOXR2 activation in the PB-FOXR2 DNA methylation group showed a 3-year survival of 0% despite multimodal therapy. The one medulloblastoma patient in the cohort is alive three years post-diagnosis.

Conclusions

FOXR2-overexpressing CNS tumors manifest significant histological, molecular, and clinical diversity and can occur as post-radiation tumors. Glioma histology and concomitant genetic alterations correlate with treatment resistance and inferior prognosis while FOXR2-overexpressing embryonal tumors show more heterogeneous outcomes. Integrating histologic and molecular diagnostic approaches is imperative for accurate prognostication and optimal therapeutic decision-making.

46
Incidence of CDKN2A/B loss and correlation of MTAP immunohistochemistry in histological grade 1 and grade 2 meningiomas

Background

While meningioma grading was historically based on histology, the 2021 WHO classification of central nervous system (CNS) tumors added two grade 3 defining molecular alterations, CDKN2A/B hom*ozygous loss and TERT promoter mutation. Both are associated with increased recurrence rates and poor overall survival. Additionally, CDKN2A/B hemizygous loss shows similar rates of recurrence and survival outcomes to hom*ozygous loss. Assessing for CDKN2A/B alterations often requires costly testing, including chromosomal microarray or fluorescent in situ hybridization (FISH). MTAP, a protein encoded by a gene adjacent to CDKN2A/B on chromosome 9p, has been suggested as a surrogate immunohistochemical marker for CDKN2A/B alterations.

Methods

152 meningiomas were analyzed, including 22 histologically grade 1 meningiomas with atypical features (“grade 1+”) and 130 histologically grade 2 meningiomas. All tumors were evaluated for CDKN2A/B with FISH. 15 cases were evaluated for MTAP immunohistochemistry.

Results

hom*ozygous loss of CDKN2A/B was detected in 3.1% of grade 2 meningiomas (4/130), and 0% of grade 1+ tumors (0/22). Localized hemizygous loss of CDKN2A/B was seen in 10.0% of grade 2 tumors (13/130) and 9.1% of grade 1+ tumors (2/22). Hemizygous loss in the setting of monosomy 9 was present in an additional 9.2% of grade 2 tumors (12/130) and 18.2% of grade 1+ tumors (4/22). While rates of hom*ozygous deletion were low, 22.3% of grade 2 tumors and 27.3% of grade 1+ tumors showed some degree of alteration in CDKN2A/B. Immunohistochemical loss of MTAP had a 100% specificity (11/11) but low sensitivity at 25% (1/4).

Conclusions

Molecular testing of grade 1+ and grade 2 meningiomas showed a low incidence of CDKN2A/B hom*ozygous loss with a significant number of tumors showing hemizygous alterations. Results from our single-institution study suggest MTAP immunohistochemistry has low sensitivity for detecting CDKN2A/B alterations and may not be a reliable surrogate.

47
Molecular and immunophenotypical analysis of SF1-expressing PitNETs reveals clinically distinct subtypes

Background

Steroidogenic Factor 1 (SF1 or NR5A1) is a transcription factor (TF) that regulates gonadal and adrenal functions, and contributes to differentiation of gonadotroph cells in the anterior pituitary. According to the CNS WHO 5th edition, SF1 expression defines gonadotrophic lineage for PitNETs. However, SF1 can be expressed aberrantly in other lineages without concomitant expression of FSH or LH. Here, we aimed to characterize landscape of SF1-positive PitNETs.

Methods

We analyzed a cohort of 207 PitNETs operated at NYU Langone Health or Bellevue Hospital. Demographic, clinical, and pathological data was retrieved from clinical records. Tumors were analyzed by DNA methylation using Illumina EPIC array, Next Generation Sequencing (NGS) using NYU Genome PACT, and NYU Fusion SEQER. All tumors were classified using Heidelberg. DNA methylation classifier and copy number profiles assed using conumee package and visual inspection. Unsupervised hierarchical clustering was performed using the 1000 most variable methylation probes. A Chi-squared test was performed for statistical analysis.

Results

Our cohort included 116 PitNETs that expressed SF1 on IHC; 107 (92.2%) expressed SF1 only, eight (6.9%) co-expressed SF1 and Pit1, and one case (0.9%) co-expressed SF1, Pit1, and T-Pit. Clinically, SF1 expression was associated with invasion of adjacent structures (p = 0.03) and nonfunctioning status (p < 0.001) compared to non-SF1 tumors. By DNA methylation, 97% of SF1-only cases classified as PITAD_FSH_LH, whereas only 22% of the multiple TFs cases classified as PITAD_FSH_LH. Unsupervised hierarchical clustering of PitNETs separated the PITAD_FSH_LH tumors into one cluster distinct from the other lineages. This cluster showed a relatively hypomethylated profile and simple CNV patterns (p < 0.001). NGS did not show recurrent mutations in the SF1 cluster.

Conclusions

SF1 expressing PitNETs form a clinically and molecularly distinct tumor type characterized by hormonal silencing, tumor invasion, simple CNV profile, and hypomethylation. The gonadotrophic designation may not be clinically informative since they are all nonfunctioning.

48
Rethinking Diagnostic Specificity: NKX2.2/CD99 Co-expression in Embryonal Tumor with Multilayered Rosettes

Background

NKX2.2 and CD99 co-expression by immunohistochemistry (IHC) is considered highly specific for Ewing sarcoma (ES), but to our knowledge, its specificity in the central nervous system (CNS) is not well-established. Recently, we encountered a case of a dural metastasis lacking characteristic rosettes and exhibiting NKX2.2/CD99 co-expression with only patchy LIN28A staining. Molecular studies, including methylation profiling, ultimately confirmed the diagnosis as Embryonal Tumor with Multilayered Rosettes (ETMR). This prompted us to investigate the prevalence of this unexpected marker combination in ETMR.

Methods

We analyzed NKX2.2, CD99, and LIN28A expression in 10 ETMR cases from 8 patients. Two independent pathologists evaluated staining intensity and percentage of positive cells.

Results

All (10/10, 100%) ETMR cases showed at least focal co-expression of NKX2.2 and CD99. All (10/10, 100%) cases also exhibited at least focal LIN28A expression.

Conclusions

NKX2.2/CD99 co-expression was consistently observed in our ETMR cohort, challenging its high specificity for ES. Particularly in the clinical context of dural metastasis, this pattern of immunoreactivity raises the potential for misdiagnosis as ES. Our findings underscore the importance of integrating all available clinical, pathological, and molecular data for accurate diagnosis. Further research with larger and more diverse CNS embryonal tumor cohorts is warranted to validate these observations and refine the diagnostic roles of NKX2.2, CD99, and LIN28A in CNS tumors.

PLATFORM 7: Developmental/Pediatric
49
Building a gene expression atlas of developing brainstem motor neurons to study rare congenital neurologic disorders

Background

Ocular motor neurons (OMNs) in the brainstem mediate eye movement and are differentially affected in some rare congenital neurologic diseases. In cases such as Duane Syndrome, specific OMN subpopulations show disrupted or aberrant innervation while other subpopulations remain unaffected, but mechanisms underlying differential susceptibilities have not yet been identified. Here we generate a transcriptomic atlas to analyze unique gene expression patterns of each developing MN type as a toolbox to help study these disorders.

Methods

We combined multiple mouse genetic reporter lines with intersectional temporal (embryonic days E9.5 to E18.5) and spatial transcriptomics (single cell/nuclei RNA-seq, and Slide-Seq) to isolate and compare eight distinct mouse MN populations: the three oculomotor nuclei (CN3, CN4, CN6) and the other primary MN types (CN5, CN7, CN9/10, CN12 and spinal MNs). Sample integration posed a significant “batch effect” challenge since different types of samples were acquired via multiple methods over many ages. We compared multiple benchmarked high quality integration pipelines to balance batch correction vs. bioconservation. Once cells were identified and labeled we used scDREAMER-SUP, a semi-supervised deep learning algorithm, using cell label annotations as a means to achieve further bioconservation and batch correction. We built a visualization tool to investigate integration quality. Cell clusters were mapped onto spatial transcriptomic slide-seq samples from E11.5 and E14.5 and compared with developmental mouse atlases to confirm cell identities.

Results

Candidate marker genes of each cell population were further validated via database analysis and RNA in situ hybridization. We successfully integrated a developmental time course of mouse MN gene expression from disparate sample types. The resulting atlas can be transposed to label and identify spatial structures within slide-seq datasets to identify cells in time and space.

Conclusions

Overall, this atlas uncovers distinct developmental gene expression patterns and provides new tools to study their differential vulnerability in motor neuron related disorders.

50
Disrupted rhombic lip developmental in medulloblastoma oncogenesis: Taking another look at cerebellar dysplasia & heterotopia

Background

Group 3 and 4 Medulloblastoma (MB) oncogenesis is poorly understood. Recent studies elucidated human rhombic lip cellular populations susceptible to abnormal proliferation and molecular alterations leading to MB formation. We aimed to describe the prevalence of abnormal cellular populations in the rhombic lip across a developmental spectrum via histologic evaluation of the cerebellar vermis in a postmortem cohort.

Methods

Autopsy brains submitted to the pediatric neuropathology (NP) service over a 16 month time period were assessed for the presence of intact cerebellar vermi. When evaluable, parasagittal or horizontal sections to include the nodulus were submitted and histologically reviewed for the presence of cerebellar dysplasia/heterotopia (CbllD/H). We recorded presence of CbllD/H, age, sex, NP and autopsy diagnoses including other developmental anomalies, and available genetic testing results.

Results

Twenty-eight cases had intact cerebellar vermi (age range: 25.3 gestational weeks to 27 years); 5 were intrauterine demises (age range 34.5 to 39.6 gestational weeks), and 9 liveborn infants were less than 40 weeks corrected age at time of death (age range 25.3 to 38.4 gestational weeks). Twelve cases (43%) (age range 25.3 gestational weeks to 6 months) demonstrated vermian CbllD/H and all classification subtypes of CblllD/H were represented; one had additional NP developmental abnormalities. Antemortem genetic testing was performed in 13 cases. Five of these cases had CbllD/H, 3 of which had genetic abnormalities including trisomy 18 (1) and point mutations (2).

Conclusions

As prior studies have described, we confirmed the high incidence of CbllD/H within the vermis in our autopsy cohort. Given recent studies elucidating Group 3 and 4 MB oncogenesis in relation to CbllD/H, additional studies including immunohistochemical characterization of CBllD/H is warranted.

51
Autopsy-Based Insights into Pediatric Nontraumatic Intracranial Hemorrhage: A Ten-Year Retrospective Review

Background

Nontraumatic intracranial hemorrhage (NICH) represents a critical yet understudied pathology in pediatric populations, particularly in the context of hospital autopsies. Existing research predominantly concentrates on radiologic and clinical data, leaving gross and microscopic neuropathologic correlates less explored. This study aims to offer insights into NICH, thereby enhancing our grasp of its broader implications on pediatric health outcomes.

Methods

An IRB-approved 10-year retrospective analysis of autopsies conducted at Children’s Hospital Los Angeles from 2014 to 2023 was performed. Inclusion criteria included intracranial hemorrhage, no history of head trauma, and a complete, unrestricted autopsy. Associations were evaluated utilizing the Fisher’s Exact Test.

Results

Analysis of 348 autopsies identified 130 cases of NICH (37%). The distribution across different age groups included: 5 fetuses, 46 neonates, 41 infants, 17 children, 8 early adolescents (ages 11-14), 4 middle adolescents (ages 15-17), and 9 late adolescents (ages 18-21). There were 76 males and 54 females. Hemorrhage subtypes included subarachnoid in 61 (47%), parenchymal in 53 (41%), subdural in 39 (30%), interventricular in 38 (29%), germinal matrix in 16 (12%), and epidural in two cases (1.5%) both of which were associated with recent external ventricular drainage. Notably, 17 cases (13%) demonstrated microscopic hemorrhage without gross findings. Associated conditions spanned oncologic, neonatal, developmental, genetic, infectious, and hematologic categories. A vascular malformation was observed in only one case.

Conclusions

Our study identifies NICH in 37% of pediatric autopsies over a decade. While vascular malformations are reportedly a common cause of intracranial hemorrhage, our cohort included only one. Developmental, infectious, and congenital associations were more frequent. Analysis accounting for hemorrhage subtype distribution revealed a proportionally higher incidence of subdural hemorrhage in early adolescents, but further studies are warranted. Collectively, the findings reinforce the importance of autopsy studies in elucidating the complex mechanisms underlying NICH.

52
Biomechanical Contributions of Tensegrity in the Formation of Polymicrogyria

Background

Polymicrogyria is a malformation of cortical development involving complex cortical ribbon undulations with loss of normal laminar architecture. This anomaly is etiologically diverse, with congenital cytomegalovirus encephalitis (cCMVE), genetic (Walker-Warburg syndrome, Zellweger syndrome, COL4A1-mutation, etc.), and vascular/ischemic phenomena at a variety of gestational stages all capable of producing morphologically stereotyped lesions, leading some to favour disruption of structural/biomechanical forces as a unifying mechanism. Tensegrity provides a means of explaining both the complex pattern of normal gyrification and its disruption in polymicrogyria by evoking the interplay between tensile and compressive forces at the cellular and macromolecular level.

Methods

A collection of cCMVE-associated polymicrogyria cases was examined, with gestational ages ranging from 16-39 weeks. These cases were compared to age-matched controls without findings of polymicrogyria. To investigate the possible contributions of tensile forces imparted by neuronal and glial processes, cases were evaluated for relative depletion of projection neuron populations, neuronal and glial process disruption, pial-glial membrane injury, and leptomeningeal heterotopias.

Results

Clinical details and pathological findings indicate that the responsible insults occurred before neuronal migration was complete, lasted several weeks, and led to a variety of structural perturbations to the developing brain. In the affected cases, several findings were almost invariable: subventricular zone necrosis, subventricular germinal cell infection, radial glial disruption, and injuries to the pial-glial border. Subcortical neuronal and leptomeningeal heterotopias were also common.

Conclusions

These cases add to the evidence that polymicrogyria is not solely post-migrational and that a variety of injuries (and timings) can replicate its relatively stereotyped morphology. Furthermore, the associated disruption of projection neurons, radial glia, and pial-glial interface – elements capable of exerting tensile and compressive forces that might influence normal gyrification – provides a biomechanical basis for polymicrogyria formation under the framework of tensegrity.

53
Frequent Cerebral Intraparenchymal Calcification in Cases of Sudden Unexplained Death in Pediatrics

Background

Sudden unexplained death in childhood (SUDC) is a diagnosis of exclusion after complete and thorough death investigation. As a leading cause of death in children, SUDC accounts for more deaths than cardiac disease and cancer. A proposed cause of SUDC is believed to be an underlying disease state accompanied by an environmental stressor. The “underlying disease state”, however, is currently unknown.

Methods

Postmortem examination of the brain was performed on 32 cases of SUDC (Male:Female = 1.67:1; age at death: 0 day-3.5 years [median= 3.5 months]) and 37 cases of age-matched controls (Male:Female = 1.18:1; age at death: 0 day-4.5 years [median= 3.2 months]). Those with evidence of co-sleeping or asphyxiation were designated as the control group.

Results

The brains examined were predominantly grossly unremarkable, with the exception of three cases (9.4%; 3/32) including one Down syndrome case with microcephaly. We identified histopathologic findings in 56.25% (18/32) of the SUDC and 10.81% (4/37) of the control cases in our cohort. The histologic findings seen in SUDC cases included: microcalcification (21.9%; 7/32), acute hypoxic/ischemic changes (12.5%; 4/32, periventricular leukomalacia (9.4%; 3/32), granule cell dispersion (6.25%; 2/32), reactive gliosis (3.13%; 1/32), and hippocampal sclerosis (3.13%; 1/32). In comparison, only 2.7% (1/37) of control cases showed microcalcifications in the brain. The microcalcifications were noted in the subcortical white matter in a perivascular distribution and often associated with microbleeds. They may represent neuronal ferrugination.

Conclusions

Subcortical microcalcification is a frequent finding in SUDC. The etiology and clinical significance are unclear. However, calcification represents findings of chronic or remote injury, which may result from recurrent hypoxic/ischemic insults that predisposes young children to sudden death. This is supported by the relative dearth of findings in our control group.

54
Congenital Moebius Syndrome Shows Primary Abducens Motor Nuclei Lesion, Secondary Facial Neuron Loss, and Abnormal Tracts

Background

Moebius syndrome (MBS) is a rare congenital condition characterized by facial and horizontal gaze palsies, likely due to the absence or underdevelopment of the abducens (CN6) and facial (CN7) cranial nerve nuclei. Proposed etiologies include vascular injury and genetic factors. We utilized histology, MRI/DTI imaging, and whole genome sequencing to investigate MBS.

Methods

We evaluated postmortem brainstems from two adults with MBS against controls. MRI was used for structural evaluation and tract tracing. Histological analysis involved staining for cell morphology, nerve tracts, and a panel of markers. Genomic analysis was conducted on blood and brainstem tissue from the first individual (I1), and on blood from the second individual (I2) and their parents, to identify germline and somatic mutations.

Results

CN6 and CN7 were notably absent in I1 and I2, with bilateral calcified and iron-deposit lesions noted near where CN6 should exist. In I1, CN6 motor neurons were entirely absent, and CN7 motor neurons were largely depleted with gliosis in the surrounding region. Bilateral abnormal dorsal-ventral tracts were identified near the expected paths of CN6, but turned rostrally rather than exiting the brainstem, and were negative for acetylcholinesterase. No definitive candidate genes emerged from genomic analyses.

Conclusions

Our findings suggest initial damage to the dorsal medulla/pons embryonically damages CN6—with secondary CN7 motor neuron degeneration. Pathways involved in abducens/facial control may contribute to the abnormal tracts. MBS may be linked to hypoperfusion resulting from critical developmental vulnerabilities around 6 weeks of gestation, when there’s substantial brainstem and vascular remodeling. Our study offers insights into the pathophysiology of MBS-associated cranial nerve anomalies and in the absence of a genetic etiology, underscores a potential vascular cause, possibly linked to early gestation vascular perfusion changes.

55
Keratan Sulfate: Chemical Template for Neuroblast Migratory Pathways and Axonal Fascicles

Background

Keratan sulfate (KS) is a proteoglycan secreted in the fetal brain by astrocytes and radial glia into extracellular parenchyma as granulo-filamentous deposits. KS surrounds neurons except dendritic spines, repelling glutamatergic and facilitating GABAergic axons. The same genes are expressed in both neuroblast migration and axonal growth. This study examines the role of KS in morphogenesis of the normally developing human fetal forebrain.

Methods

28 normal human fetal brains from 9 to 42 weeks gestational age (wk GA) were studied at autopsy. KS was examined by immunocytochemical reactivity in formalin-fixed paraffin-embedded sections, plus various other cellular markers including NeuN, MAP2, synaptophysin, vimentin, nestin, S100β protein.

Results

Radial and tangential neuroblast migratory pathways from subventricular zone to cortical plate were marked by KS deposits as early as 9wk GA, shortly after neuroblast migration was initiated. During later gestation this reactivity gradually diminished and disappeared by term. Long axonal fascicles of the internal capsule and short fascicles of intrinsic bundles of globus pallidus and corpus striatum also appeared as early as 9-12wk, as fascicular sleeves before axons even entered such fascicles. Intense KS occurs in astrocytic cytoplasm and extracellular parenchyma at 9wk in globus pallidus, 15wk thalamus, 18wk corpus striatum, 22wk cortical plate, hippocampus postnatally. Corpus callosum, and anterior commissure do not exhibit KS at any age. Optic chiasm exhibits reactivity peripherally but not around internal sub-fascicles.

Conclusions

KS forms axonal fascicles before axons enter and is transitory along neuroblast migratory pathways to create a chemical template (not structural scaffold) for axonal trajectories and cellular migration during morphogenesis.

56
Multimodal high-resolution whole-brain characterization of holoprosencephaly (HPE) from 13-22 gestational weeks

Background

Holoprosencephaly (HPE) is a commonly occurring patterning disorder of the forebrain, with a wide phenotypic spectrum of dysmorphism of the face and brain. While the onset of the induction defect is well known to be an early embryonic event (classically affecting the sonic hedgehog pathway), the most detailed neuropathology reports are from third-trimester fetuses, infants or children. We hypothesized that comprehensive analyses from the second trimester would assist in understanding the cellular pathogenesis of the eventual final phenotype.

Methods

We employed a novel, comprehensive multimodal approach, including prenatal ultrasonography, postmortem MRI, whole-brain embedding, with blockface imaging of serial sections, followed by three-dimensional (3D) reconstruction of the digitized whole-mount slides, to characterize 3 HPE brains from 13-22 gestational weeks (GW), comparing them to identically processed age-matched controls.

Results

Aside from the classical failure of cleavage (midline fusion) of the rostral forebrain, our HPE cases showed: 1) established rostral subarachnoid glioneuronal (marginal) heterotopia; 2) thinning of the cerebral mantle posterolaterally, but with an “evenly cellular” cortical plate, conspicuously lacking “glomeruloid” structures as reported previously in more mature cases; 3) “reactive”-appearing astrocytes on GFAP immunostaining of the cortical plate; 4) clustering of migrating cells in posterolateral subcortical cell layers suggesting “incipient” laminar heterotopia, visible even at 13GW; and 5) lateral and horizontal displacement of the hippocampus on 3D segmentation, despite a relatively normal laminar arrangement of cells.

Conclusions

Our approach utilizing multiscale, multimodal 3D analytics provides a complementary perspective on the timing and evolution of the neuropathology of HPE in the second trimester.

PLATFORM 8: Infectious, Demyelinating and Inflammatory, Peripheral Nerve/Muscle, Ophthalmic Pathology
57
Multifocal Necrotizing Leukoencephalopathy with a Pontine Predilection Following BCMA CAR-T in a Critically Ill Patient

Background

The emergence of CAR-T therapy has introduced new neurological complications, notably the clinical syndrome Immune effector Cell-Associated Neurotoxicity Syndrome (ICANS). However, the neuropathology of CAR-T neurotoxicity is not well characterized. Despite increasing clinical classification, the mechanisms and associated neuropathological findings of CAR-T neurotoxicity remain underexplored. Multifocal necrotizing leukoencephalopathy with pontine predilection (MNL-PP) is a neuropathological finding associated with profound immunosuppression and is characterized by microscopic foci of necrosis confined to the white matter of the basis pontis. MNL-PP has previously been associated with congenital and acquired immunodeficiency, chemotherapy, and CD19-directed CAR-T therapy, among other causes. In this case study, we describe the neuropathological findings of a patient with multiple myeloma who received BCMA-directed CAR-T.

Methods

We identified a case of a 57-year-old male patient who received BCMA-directed CAR-T, 6 years after his original diagnosis. Following infusion, he developed cytokine release syndrome (CRS) and immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS), from which he initially recovered. The patient presented one week later in septic shock with bacteremia, viremia, and CAR-T induced aplastic anemia with recurrent/refractory IEC-HS. Despite autologous stem cell boost, the patient continued to decline and ultimately passed.

Results

On autopsy, the patient displayed multi-organ ischemic necrosis involving most visceral organs and findings consistent with aspiration pneumonia. Neuropathological evaluation demonstrated diffuse and marked CD163-positive cells, abundant astrogliosis, and mildly expanded perivascular spaces. The basis pontis showed multiple small foci of white matter spongiosis and necrosis with associated demyelination, patchy axonal loss, and b-APP axonal spheroids.

Conclusions

We describe the postmortem neuropathological findings of a complex and critically ill patient with multiple myeloma who received BCMA-directed CAR-T and was found to have evidence of MNL-PP on postmortem evaluation. Although not directly attributable to CAR-T treatment, we believe this report contributes to the ongoing investigation into the neuropathological findings seen following CAR-T.

58
Neuropathologic findings in a community-based autopsy cohort of older, virally-suppressed, people with HIV

Background

Combination antiretroviral therapy (cART) has reduced the incidence of HIV-related mortality. Consequently, there is a growing population of older, virally-suppressed, people with HIV (PWH). cART has also decreased the prevalence of HIV-associated dementia. However, many PWH experience milder forms of neurocognitive impairment. It is unclear what pathology, if any, is associated with this phenotype and whether this aging population of PWH demonstrates distinct patterns of neurodegenerative pathology compared to uninfected individuals.

Methods

We performed a community-based case-control study where we examined brain tissue from autopsies performed at the Johns Hopkins Hospital between 2015-2023 for 13 virally-suppressed PWH and from 13 HIV-negative subjects. We characterized and performed semi-quantitative analysis of the neurodegenerative pathology, including beta-amyloid pathology, tau pathology, alpha-synuclein pathology, and TDP-43 pathology, in each group.

Results

PWH and uninfected controls had similar demographics, including age (median: 59 vs. 57 years), male sex (54%), and race (100% black). Medical co-morbidities were also similar with a trend towards a higher prevalence of HCV seropositivity in PWH (46% vs. 15%; p = 0.09). Common histologic brain findings included atherosclerosis (46% vs. 54%) and infarcts (54% vs. 38%). With respect to neurodegenerative pathology, while no statistically significant differences were found between our relatively small cohorts, PWH demonstrated trends towards increased prevalence of beta-amyloid pathology (69% vs. 54%), caudal entorhinal cortex neurofibrillary tangles (100% vs. 77%), aging-related tau astrogliopathy (23% vs. 0%), and alpha-synuclein pathology (31% vs. 8%); as well as increased severity of diffuse plaque pathology (median density score, 2 vs. 1).

Conclusions

Compared to previous studies that have relied on research study subjects, our community-based cohort of older, virally-suppressed PWH demonstrates trends towards more prevalent and more severe neurodegenerative pathology in PWH. These findings highlight the need larger community-based cohort studies as we aim to characterize and understand the mechanisms of HIV-associated neuropathology in virally-suppressed individuals.

59
Comparison of neuropathologies among rabies patients treated with standard, Milwaukee, and/or experimental AAV-RAB strategies

Background

Symptomatic rabies, treated with supportive medical care, has an essentially uniformly fatal outcome. The Milwaukee Protocol (MP; 2005) is based on the concept that, since rabies is not neuronolytic, induction of coma and prevention of excitotoxicity/seizures could allow host immune clearance of the virus and neurologic recovery. MP has had, however, no meaningful efficacy in scores of reports. Antivirals and, more recently, adenoviral vector delivery of anti-rabies virus antibody (AAV-RAB) have been applied in a few patients without improving outcome.

Methods

We compared the autopsy neuropathology of 3 symptomatic men, all receiving standard (supportive) care and additional treatment as follows: Case 1 (24yo) had a dog bite on the lip; post-exposure prophylaxis (PEP) at 0 and 7d; symptoms at 17d; Favipiravir at 20d; Rabishield intrathecal at 20 and 21d; and death at 21d. Case 2 (17yo) had an unknown exposure and no PEP; MP initiated after 4d of symptoms; death at 26d. Case 3 (57yo) suffered dog bite on leg; no PEP; symptoms at 6mo; MP begun after 6d of symptoms; Favipiravir at 9d; AAV-RAB at 11d; death at 30d. Standard histology was done for Cases 2 and 3, while Case 1 underwent multimodal analysis and 3D histology (PMID 26181725).

Results

In Case 1, inflammation was sparse and correlated with viral nucleocapsid immunopositivity, mapped incrementally from brainstem to diencephalon to cerebral cortex. Case 2 had moderate forebrain inflammation in areas of viral immunostaining. Case 3 had marked lymphoplasmacytic inflammation and neuronophagia in virtually all gray matter sites (cultivable virus recovered postmortem).

Conclusions

Gray matter inflammation was similar between antiviral- or MP-treated cases. By contrast, the MP+antiviral+experimental AAV-RAB case had much greater inflammation. While these differences could be due to rabiesvirus genotypes, host factors including age, and/or survival intervals, a pro-inflammatory effect of the experimental AAV-RAB protocol urges further analysis of such cases.

60
Histological and Immunohistochemical investigation of inflammatory cells in a geographic atrophy case

Background

Geographic atrophy (GA) is a chronic progressive degeneration of the macula, affecting millions of patients with age-related macular degeneration (AMD), severely impairing central vision. Histologically, GA is characterized by atrophy of outer retinal layer (ORL), retinal pigment epithelium (RPE) and choriocapillaris. Inflammatory damage is proposed to cause GA. However, the histological features and immunohistochemical characteristics of inflammation in human GA eyes are not well understood due to scarcity of human GA eye tissues. Here we characterized a GA eye and evaluated the expression of different inflammatory cell types and compared to normal eye.

Methods

We identified a case of a GA in the left eye collected from an 88-year-old autopsy female. Histologic examination showed focal atrophy of the ORL, RPE and choriocapillaris involving the entire macula, compatible with GA. Additional inflammatory immunohistochemical staining was conducted for CD45RB, CD20, CD3, CD138, CD68, CD163, PU.1, C3D, CD21. Results were compared with stains performed on the control right eye (with no GA).

Results

H&E sections of the left eye showed atrophy of the ORL, RPE and choriocapillaris, compatible with histopathologic diagnosis of GA. Macrophages markers CD163, CD68 and PU.1 highlighted considerable macrophages at the leading edges of the atrophy. By contrast, CD138 plasma marker cell, and CD45RB, CD3, and CD20 markers for lymphocytes were negative at the atrophy. The contralateral healthy right eye did not show positive inflammatory cell markers in the retina. Complement markers (CD21, C3D) were positive in many blood vessels walls in both eyes.

Conclusions

In our histologically-confirmed GA eye, immunohistochemistry supported involvement of macrophages at the edge of the RPE degeneration. By contrast, other inflammatory cell types did not show consistent increase. Activated complement markers labeled blood vessels throughout the choriocapillaris in both GA and healthy eyes. Our findings suggest that macrophage-mediated inflammation may be involved in RPE degeneration during GA pathogenesis.

61
Deep immunoprofiling of inclusion body myositis

Background

Sporadic inclusion body myositis (IBM), the most common idiopathic inflammatory myopathy (IIM) in patients over the age of 50, presents with asymmetric muscle weakness, predominantly involving long finger flexors and quadriceps muscle. In general, CD8+ T cell invasion of muscle tissues is a prominent histological feature of IBM, and highly differentiated cytotoxic T cells are considered to be an important regulator of IBM pathogenesis. However, the drivers of such differentiation remain unclear.

Methods

We performed a deep profiling of peripheral blood mononuclear cells in IBM patients compared to healthy controls by examining the gene expression at single cell level along with complete B cell repertoire (BCR) and T cell repertoire (TCR) analyses. We also performed spatial transcriptomics analysis of immune cell infiltrates of skeletal muscle from the IBM subjects.

Results

We included four patients with IBM, three men, and one woman, with a mean age of 71.5 ± 7.6 years and disease duration of 8 ± 4.4 years in this preliminary analysis. Two healthy controls were recruited (both men, age 47.5 ± 7.6 years). We noted differential gene expression in the IBM B cell compartment that were related to immunoglobulin production, leucocyte migration, and T-cell differentiation. As previously reported, there was a strong gene expression signature of highly differentiated cytotoxic T cells in IBM. Pseudo-time analysis reflected a distinct developmental trajectory of these T cells compared to healthy controls, which associated with a group of transcription factors (TF) including those found in the T-box family.

Conclusions

This preliminary analysis reinforces the autoimmune nature of IBM, further implicating a potential role for both B cells and uniquely developed cytotoxic T cells in the pathophysiology of IBM.

62
Comparative study on lupus myositis, dermatomyositis, immune mediated necrotizing myopathy, and antisynthetase syndrome associated myositis

Background

In a recent study, we showed that lupus myositis (LM) could present in the forms of panfascicular necrotizing myopathy (35%), perifascicular necrotizing myopathy (20%), diffuse necrotizing myopathy (20%), polymyositis (15%), and necrotizing vasculitis (10%). Those various morphologies posed a significant diagnostic challenge on muscle biopsy as they might be histologically indistinguishable from immune mediated necrotizing myopathy (IMNM), dermatomyositis (DM), or antisynthetase syndrome associated myositis (ASyS), respectively. Better immunohistochemical markers are needed to differentiate the above entities.

Methods

Myxovirus resistant protein (MxA), MHC class I (MHC1), MHC class II (MHC2) immunohistochemistry (IHC) as well as detailed histological analyses were performed on the muscle biopsies from clino-serologically proven cases of LM (n = 20), DM (n = 14), IMNM (n = 22) and ASyS (n = 15).

Results

MxA sarcoplasmic reactivity was present in 94.4% DM, 80% LM, 20% ASyS, and 0% IMNM cases. MHC1 sarcoplasmic or sarcolemmal reactivity was present in 90.1% DM, 78.5% LM, 93.3% ASyS, and 35.0% of IMNM cases. MHC1 expression was usually diffuse in LM, compared to perifascicular in DM, ASyS, and patchy/mosaic in IMNM. MHC2 sarcoplasmic reactivity was present in 14.3% DM, 84.2% LM, 100% ASyS and 9.0% for IMNM. MHC2 reactivity was strongest in ASyS and variable in LM. Histologically, mixed T and B lymphocytic inflammation was present in 70% of LM cases, indistinguishable from DM (p = 1.0), but significantly more than IMNM (p = 0.0004).

Conclusions

As a rule, a MxA+/MHC1+/MHC2- IHC profile supports DM; a MxA+/MHC1+/MHC2+ IHC profile supports LM, a MxA-/MHC1+/MHC2+ IHC profile supports ASyS; and a MxA-/MHC1-/MHC2- profile supports IMNM. However, exceptions do exist, and the pathologic findings still need to correlate with clinic-serological findings for a definitive diagnosis.

63
Complex presentation of leukoencephalopathy with brain calcifications and cysts (Labrune Syndrome): A rare case report

Background

Leukoencephalopathy with brain calcifications and cysts (LCC) also known as Labrune syndrome, is an extremely rare leukodystrophy with fewer than 100 cases reported worldwide since it was first described in 1996. It is characterized by progressive deterioration and brain calcifications and cysts, related to mutations in the SNORD118 gene with an autosomal recessive inheritance.

Methods

We present a case of a 62-year-old man with right-sided weakness, facial droop, dysarthria, urinary incontinence, and unintentional weight loss. Imaging studies revealed multiple calcified supratentorial lesions, linear calcifications, a cystic lesion and extensive vasogenic edema, an unusual constellation of findings not typical of metastatic disease, with only some similarity to CNS infection such as neurocysticercosis.

Results

Brain biopsy was performed, and histology showed classic LCC features of brain parenchyma with extensive microangiopathy, ranging from hyalinization, fibrin deposition and occlusion of the lumen. Other features included loss of myelin, perivascular hemosiderin-laden macrophages and reactive astrocytosis, including Rosenthal fibers, and scattered concentric parenchymal concretions. Next generation sequencing identified two germline heterozygous pathogenic variants in the SNORD118 gene associated with autosomal recessive leukoencephalopathy with brain calcifications and cysts.

Conclusions

Labrune syndrome poses diagnostic challenges due to its rarity, diverse clinical and radiological presentation. This case underscores the importance of integrating clinical presentation with imaging, histopathology, and genetic findings for accurate diagnosis. The presence of classic pathological findings, in conjunction with pathogenic SNORD118 variants, is diagnostic of LCC. Management remains supportive, with antiepileptic drugs and steroids as mainstays for symptom control.

64
Targeted Next-Generation Sequencing Reveals Frequent Copy Number Alterations in Intraocular Leiomyomas

Background

Leiomyomas are benign smooth muscle tumors commonly involving the uterus, soft tissue, and gastrointestinal tract, but in rare cases can also arise intraocularly. Notably, some intraocular leiomyomas demonstrate atypia, mitotic activity, and/or necrosis, worrisome for “intraocular leiomyosarcoma,” despite the lack of established criteria for malignancy. We performed targeted next-generation sequencing of a series of intraocular leiomyomas, including one with worrisome histologic findings, to investigate for potential recurrent alterations, differences between tumors in anterior versus posterior segment involvement, and possible shared alterations with leiomyomas of other sites.

Methods

We identified eight patients diagnosed with intraocular leiomyoma and performed UCSF500 targeted next-generation sequencing on six cases with sufficient material.

Results

The cohort included 4 female and 4 male patients ranging from age 16 to 70 years. Two tumors limited to the ciliary body did not yield sufficient DNA. The largest tumor in the cohort (30 mm) seen in the youngest patient fills the globe and shows suprachoroidal involvement without a distinct ciliary body attachment and shows mitotic activity (1/10 high-power fields), cytologic atypia and a small focus of necrosis. Sequencing did not reveal any pathogenic single nucleotide variants, deep deletions, amplifications or structural rearrangements involving the 529 cancer genes targeted in this assay. However, copy number analysis demonstrated multiple, recurrent whole chromosome losses (including losses of 1, 2, 3, 10, 15q, 22q).

Conclusions

Loss of numerous whole chromosomes suggestive of near haploidization of the genome is a rare cytogenetic pattern seen in Hurthle cell carcinoma of the thyroid, inflammatory rhabdomyoblastic tumor, and a subtype of mesothelioma, but not reported in leiomyomas of other sites. Intraocular leiomyomas do not harbor MED12 mutations or HGMA2 translocations seen in a subset of uterine and soft tissue leiomyomas. Cytogenetic features of the large tumor with atypia and mitotic activity did not differ from the rest of the cohort.

Poster Presentations #1 – #264
Posters: Demyelinating/Inflammatory
1
Diverse HLA-DR-Immunopositive Cell Morphology in Multiple Sclerosis Lesions and Implications for Pathogenesis

Background

Multiple sclerosis (MS) features white matter demyelinating plaques categorized as active, chronic active, or chronic inactive based on the degree of demyelination, and inflammatory response, with participation of microglia/macrophages being pivotal. This study investigates the morphological traits of HLA-DR-immunopositive cells within these plaques to advance our comprehension of MS plaque evolution.

Methods

We conducted a comprehensive analysis of 90 plaques from six MS cases, employing various staining techniques (HE-LFB, HLA-DR, GFAP, APP, and NFP). Among these, 77 were classified into three main categories: 30 active, 33 chronic active, and 14 chronic inactive. Additionally, we examined five white matter lesions with vacuolation, two with axonal degeneration, and six challenging lesions. Six control cases were included to assess HLA-DR-immunopositive cell expression across different age groups. These cells were classified based on shape and processes into two categories: round cells without processes (macrophages) and cells with varying shapes and lengths of processes (ramified microglia).

Results

A mixture of macrophages and ramified microglia is present in all lesions, with our primary focus on characterizing the predominant cell type. Among the 30 active plaques, macrophages predominated in 22, while ramified microglia prevailed in 8 plaques. In the center of the 47 chronic plaques, scattered ramified microglia were present in 46 plaques, with one plaque featuring macrophages. Within the 33 chronic active lesions, 31 exhibited ramified microglia in the periphery, while two of them had predominantly macrophages. The intermediate zone within these plaques had a comparable proportion of HLA-DR-immunopositive cells but showed intermediate cellularity between the central and peripheral regions.

Conclusions

The predominance of macrophages in acute lesions is aligned with the phagocytic function in active demyelination. The prevalence of ramified microglia in the periphery of chronic active lesions suggests its potential protective effect in impeding demyelination.

2
Histopathological features of antemortem Marchiafava-Bignami disease with clinical and radiologic correlations

Background

Marchiafava-Bignami disease (MBD) is thought to be an extremely rare disorder characterized by corpus callosum demyelination and necrosis in the setting of chronic alcoholism, although this damage may also occur with severe malnutrition. To our knowledge there is only one antemortem case report describing the histopathological features.

Methods

Here we describe the clinical, serial radiologic, and histopathologic features of two cases of corpus callosum demyelination identified on biopsy, the first related to chronic alcoholism and the second in the setting of severe malnutrition.

Results

Brain MRI performed on a 53-year-old male with a past medical history of chronic alcoholism, heart failure, and recent development of a right facial droop revealed an expansile T2 signal abnormality with corresponding restricted diffusion and contrast enhancement involving a well-defined region of the body and isthmus of the corpus callosum. A biopsy demonstrated frequent macrophages with myelin debris in their cytoplasm and axonal loss in the area with the densest collection of macrophages. However there was still a degree of axon preservation compared to the degree of myelin loss. The second case is a 46-year-old female with end stage renal disease, severe malnutrition, and pneumonia with pleural effusion. Her hospital course was complicated by encephalopathy and quadriplegia. Brain MRI showed diffuse hyperintense signal, restricted diffusion and patchy contrast enhancement involving the entire corpus callosum and multiple bilateral foci of restricted diffusion in the centrum semiovale and periatrial white matter. Microscopy showed infiltration of sheets of macrophages with foamy cytoplasm and confluent areas of partial demyelination. Prominent myelin debris was observed in the cytoplasm of many of the macrophages with relative preservation of axons.

Conclusions

These two cases with corpus callosum lesions showing demyelination described in the setting of chronic alcoholism and severe malnutrition underscore the diverse etiologic and clinicopathologic features of MBD.

3
Granulomatosis with Polyangiitis with Frontal Cerebral Involvement

Background

Introduction: Granulomatosis with polyangiitis (GPA; formerly Wegener’s Granulomatosis) is a necrotizing, granulomatous vasculitis which affects small- to medium-size vessels, and typically presents in older men with respiratory and/or renal involvement. Cerebral involvement, resulting in neurologic manifestations such as meningitis- or stroke-like symptoms, and psychiatric disturbances such as depression, occur in < 10%.

Methods

Case history: We encountered an unanticipated diagnosis of GPA at autopsy in a 37-year-old man with a history of cocaine use and unspecified remote sinus surgery. He worked as an electrician and was found on the sidewalk outside a job site in a multi-story building. He was not known to have psychiatric illness nor suicidal ideation.

Results

Along with extensive blunt force injuries of the torso, extremities and head, external examination showed a depressed nasal bridge (“saddle nose deformity”). Internally, purulence of the frontoethmoid sinuses and erosion of the cribriform plate were associated with marked dural nodularity and thickening, and underlying encephalomalacia of both frontal lobes. Histopathology revealed typical features of GPA (multifocal vascular fibrinoid necrosis, acute and chronic inflammation, including granulomas with giant cells), extending rostrally from the frontoethmoid sinuses, through the dura and leptomeninges, and into the orbitofrontal cerebral cortex and white matter. Trichrome, elastic-van Gieson, reticulin, and PAS stains confirmed the vasculocentric inflammation. Fragments of foreign material were also identified, likely secondary to ongoing intranasal cocaine but also because of direct communication with the oropharynx. There was no evidence of renal involvement; however, pulmonary vasculature had focal perivascular chronic inflammation and fibrosis involving small vessels with associated elastin fragmentation. The cause of death was multiple blunt impact injuries, and manner undetermined (given uncertainty about the scene circ*mstances).

Conclusions

Conclusion: GPA can present unexpectedly, evade detection without full autopsy examination, and, remarkably, may cause extensive frontal lobe damage.

4
Rheumatoid Meningitis: 4 Cases with Emphasis on Possible Links to Alzheimer disease

Background

Rheumatoid meningitis (RM) is rare and presents with sufficiently wide-ranging, but non-specific, symptoms (seizures, headaches and cranial nerve palsies) and magnetic resonance imaging (MRI) features of meningeal thickening that is indistinguishable from subacute infectious meningitis. In addition, cognitive alterations due to the systemic inflammation, possibly without structural correlates within the CNS, also are known to occur. Serological testing for rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) are helpful clues for diagnosis, but are not invariably present. Thus, meningeal biopsy may be mandatory for diagnosis. Histological findings for RM commonly include leptomeningitis and small vessel vasculitis, with rheumatoid nodules and pachymeningitis being far less common. We reviewed our experience with RM biopsies, placing them into the context of cases in the literature. Unlike older prior studies, we took into account new studies that show possible links between RM and development of Alzheimer disease (AD), further assessing our cases for amyloid and tau.

Methods

Database searches for the years 2013-2023 to generate cases, coupled with chart review.

Results

4 RM cases identified: 2 male: 2 female, ages 62-79 years, (median 74 years, mean, 72 years). All patients had leptomeningeal enhancement by MRI and 2 had negative RF serology prior to meningeal biopsy. All 4 leptomeningeal/superficial cortical biopsies revealed diffuse chronic inflammation with patchy distribution of plasma cells, giant cells, vasculitis, and necrotizing rheumatoid nodules. Stains (GMS, Gram, Fite) for microorganisms and IgG4 were all negative. 2 of 4 cases had AD neuropathic changes by amyloid and tau immunostaining, 1 of which developed clinical AD 5 years post biopsy; the second patient was lost to follow-up. The remaining 2 cases had few AD changes on biopsy and remain free of clinically-diagnosed AD at 62 and 54 months post-surgery.

Conclusions

RM is a rare diagnosis with a histologic overlap with AD in a subset of cases.

5
Diffuse lymphocytic encephalomyelitis with stiff-person-like syndrome in multiple myeloma after Teclistamab treatment

Background

Subacute encephalomyelitis is a complex neurological condition with diverse etiologies, including autoimmune disorders and paraneoplastic syndromes. Although neurological complications such as progressive multifocal leukoencephalopathy has been documented in multiple myeloma (MM), motor neuron disease mimicking stiff-person syndrome has been rarely reported in MM.

Methods

We herein report a 45-year-old man with a 13-year history of IgD lambda MM who underwent allogeneic stem cell transplantation, followed by treatment with Teclistamab, a novel BCMA-targeted bispecific T cell engager. Two years after the initiation of Teclistamab, patient developed gait instability, spasticity, and upper motor neuron-pattern weakness, complicated by chronic lymphocytic meningitis. Anti-GAD65 antibody was mildly elevated in the serum but negative in CSF. Despite a clinical suspicion of stiff-person syndrome, he did not respond to immunotherapies including steroids, rituximab, cyclophosphamide, and methotrexate. The symptoms progressed even after discontinuation of Teclistamab, and he passed away eight months after the onset of these symptoms.

Results

Autopsy revealed diffuse lymphocytic encephalomyelitis with CD8>CD4 T-cell predominance across the brain and spinal cord. The T cells infiltrate all grey matter structures with significant perineuronal aggregates associated with moderate macrophage influx and microglial activation resulting in diffuse neuronal loss and gliosis. Subcortical and periventricular white matter also showed T cell infiltrates with less intensity associated with axonal injury, without definitive evidence of demyelination process. Wallerian degeneration was evident in spinal tracts, highlighting extensive axonal injury. No histology or immunohistochemistry evidence for infectious etiologies were detected. The T-cell receptor gamma gene rearrangement study revealed no neoplastic clonality in the T-cells.

Conclusions

His stiff-person-like symptoms, with low serum GAD65 and negative CSF results, suggest that immunosuppression from MM treatment may mask typical autoimmune markers, complicating diagnosis of stiff-person syndrome. Moreover, Teclistamab-associated encephalitis cannot be ruled out. Further investigation into the complex complications associated with MM treatment is crucial for understanding of its impact.

6
Calcium pyrophosphate deposition (CPPD) of the Spine: An Underrecognized Debilitating Disease

Background

Calcium pyrophosphate deposition (CPPD) disease is an inflammatory arthritis common in elderly patients. Spinal CPPD however is uncommon and presentation can vary widely as osteoarthritis, chronic inflammatory arthritis, acute pseudogout, or other rheumatologic diseases. Radiographic mimics (e.g. cystic masses, spinal infections, etc.) also pose diagnostic challenges. Accurate pathologic diagnosis is key to improve disease identification and management.

Methods

Between 2014 and 2024, we identified 10 cases of spinal CPPD. Clinical notes, imaging, operative notes, and pathology were reviewed.

Results

We identified 10 patients (age range: 63 to 85 y; 5 M, 5 F), with lumbar (9) and thoracic (1) spinal CPPD, of which 90% presented chronically and 10% acutely, all with pain, 4 with prior instrumentation, and 1 with prior trauma. CPPD was not clinically suspected in any case. Based on imaging, central canal stenosis was suspected in 8 cases followed by neuroforaminal narrowing (5/10), septic arthropathy (4/10), synovial cyst (3/10), degeneration (2/10), and epidural mass/abscess (2/10). Intraoperatively spinal cyst was described most often. Pathology showed purple granular material with anisotropic positively-birefringent rhomboid crystals embedded within fibrocartilage (10/10) with degenerative changes seen in half of cases (5/10). Two patients were treated for CPPDand 2 patients were given antibiotics. Two patients had poor outcomes, with one patient showing CPPD in a wrist joint aspirate 4 years after initial spine pathology revealed crystals.

Conclusions

Spinal CPPD is underdiagnosed and undertreated.Crystal arthropathy was not suspected in any patient that we reviewed, and CPPD was treated in only 2 cases. Variable clinical presentations can be challenging, underscoring the importance of wider recognition of this rare disease among neuropathologists. Improved identification of spinal CPPD, especially in elderly patients with back pain, and/or prior trauma/instrumentation, will promote appropriate therapy and management.

7
Incidental finding of macrophagic myofasciitis in a muscle biopsy with duch*enne muscular dystrophy

Background

We present the case of an 18-month-old boy who presented with gross motor delay, hypotonia, generalized weakness, myotonic facies, and areflexia. Creatine kinase level was 35,110 U/L. Genetic test revealed an in-frame deletion of exon 5-13 in the duch*enne muscular dystrophy (DMD) gene. Deletions in the DMD gene give rise to both duch*enne and Becker muscular dystrophies. The clinical severity depends on the disruption or maintenance of reading frames. However, there are exceptions to this rule. Deletions in the 5’ region of the DMD gene are frequently associated with unpredictable phenotypes. Muscle biopsy of the quadriceps was performed to evaluate the expression of dystrophin protein. The muscle showed moderate fiber size variation with abundant degenerating and regenerating myofibers. Immunohistochemistry demonstrated loss of expression of dystrophin epitopes (rod domain and amino terminus) and attenuated expression of dystrophin carboxy terminus and alpha-sarcoglycan, which supported the diagnosis of duch*enne muscular dystrophy. The in-frame deletion of exon 5-13 might result in the severe phenotype; this information should be considered when counselling cases with such mutations.

Methods

Microscopy

Results

Another unusual finding in this muscle biopsy was extensive lymphoid aggregates and multifocal macrophage-rich inflammation. Extensive inflammation is not a feature of DMD, although mild inflammation is not uncommon. GMS and AFB stains were negative for microorganisms. The morphologic and immunohistochemical findings ruled out lymphoproliferative or histiocytic disorders. Morin stain demonstrated blue-green, granular cytoplasmic fluorescence in the infiltrating macrophages. EM confirmed the existence of the dense spicule-like inclusions within the macrophages. The diagnosis of macrophagic myofasciitis (MMF) was made. MMF is an inflammatory condition associated with intramuscular injection of aluminum adjuvant-containing vaccines. Histologically, it’s characterized by aggregates of macrophages with abundant basophilic, PAS-positive, diastase-resistant granules without myofiber damage. The definitive diagnosis of MMF requires demonstration of aluminum within these macrophages.

Conclusions

This highlights a rare case of MMF.

Posters: Developmental/Pediatric
8
A case of meningioangiomatosis with molecular characterization

Background

A rare hamartomatous proliferation, meningioangiomatosis (MA) often presents with seizure and headache in children and young adults. MA can occur either in isolation, in association with Neurofibromatosis type 2, or in conjunction with meningioma, and may be considered neoplastic in the latter instance.

Methods

Case presentation: A 21-year-old female with no significant medical history developed intermittent numbness involving the tongue and hands. Her symptoms progressed to facial and hand twitching, and later two generalized tonic-clonic seizures. Magnetic resonance imaging showed a 2.0 cm T2/FLAIR hyperintensity involving the juxtacortical left lateral frontal lobe. She underwent left frontotemporal awake craniotomy for resection of suspected low-grade glial neoplasm.

Results

Pathology: Frozen section demonstrated cohesive spindle cell process. Permanent sections showed a meningothelial process extending along perivascular spaces. The meningothelial cells had ovoid nuclei, open chromatin, and eosinophilic cytoplasm with ill-defined borders. These cells showed patchy reactivity for progesterone receptor (PR), but were negative for EMA and glial/neuronal markers, and had low Ki67 (< 1%). No evidence of a glial or glioneuronal neoplasm or cortical dysplasia was found. No distinct meningioma was identified, and the surgeon reported that the lesion was deep from the cortical surface. Single nucleotide polymorphism (SNP) microarray found multiple chromosomal abnormalities including loss of a portion of 1p, gain of 1q, gain of a portion of 9q, and loss of a portion of 22q (including NF2).

Conclusions

SNP array demonstrates a clonal process with a possible molecular driver, as 22q loss of heterozygosity is a common driver of meningiomas. Although clonal proliferations are often considered neoplastic, somatic mutations have also been shown to underlie some vascular malformations. Thus defining MA as hamartoma versus neoplasm can be challenging. In the absence of a definite meningioma component, this lesion with NF2 loss of heterozygosity was favored to represent a non-neoplastic process.

9
Comprehensive analysis of MYB/MYBL1-altered pediatric-type diffuse low-grade glioma

Background

Pediatric-type diffuse low-grade gliomas (pLGG) harboring recurrent alterations involving MYB or MYBL1 are closely related tumors. Detailed treatment and outcome data of large cohorts are still limited. This study aimed to comprehensively evaluate pLGG with these alterations to define optimal therapeutic strategies.

Methods

We retrospectively reviewed details of pLGG with MYB or MYBL1 alterations from patients treated or referred for pathologic review at St. Jude Children’s Research Hospital. Tumor specimens were centrally reviewed, and clinical data were collated.

Results

Thirty-three patients (18 male; median age 5 years, range 1.8-40 years) were identified. Two tumors had MYBL1 alterations; 31 had MYB alterations, MYB::QKI fusion being the most common (n = 10, 30%). No other genetic alterations were identified in any of the tumors. Most tumors were in the cerebral hemispheres (n = 22, 67%). Two patients (6%) had metastasis at diagnosis. Twenty-three (70%) tumors were diagnosed as angiocentric glioma based on their dominant growth patterns. Five tumors (15%) were diagnosed as diffuse astrocytoma, and 5 (15%) as isomorphic diffuse glioma. The median follow-up was 6.1 years. The 5-year event-free survival (EFS) rate was 81.3±8.3%; the 5-year overall survival (OS) rate was 96.4±4.1%. Patients receiving a near-total or gross-total resection had a 5-year EFS of 100%; those receiving a biopsy or subtotal resection had a 5-year EFS rate of 56.6±15.2% (p < 0.01). No difference in EFS was observed based on location, histology, or molecular alterations. No histologic evidence of transformation was noted in any subsequent or recurrent samples. However, the tumors that progressed or metastasized may have distinct methylation profiles with evidence of activation of the MAPK and PI3K/AKT/mTOR pathways.

Conclusions

pLGG with MYB or MYBL1 alterations have good outcomes. Our findings suggest that surgical resectability is a crucial determinant of EFS. Further characterization is required to identify optimal treatment strategies for metastatic or progressive tumors.

10
Focal Cortical Dysplasia in Proximity to Arteriovenous Malformation: Report of Four Cases and Review of the Literature

Background

Central nervous system arteriovenous malformations (AVMs) and focal cortical dysplasia (FCD) are congenital lesions originating during fetal development. We present four cases in which FCD was identified adjacent to a surgically resected AVM.

Methods

Clinical and histopathologic features were summarized. A literature search was conducted.

Results

Resection of a clinically diagnosed AVM was performed on four patients aged 25-39 years (two men and two women). Two patients had a history of seizures, with onset ranging from three years before resection to onset at the time of presentation. One patient lacked a seizure history. Resection was undertaken and FCD was identified in intact cortex in proximity to the resected AVM. In each case persistent, radial columns or laminae of neurons (lamination ), neurons showing evidence of granular cell and pyramidal cell maturation but lacking horizontal migration in the cortex, consistent with designation as International League Against Epilepsy FCD type Ia were observed in proximity to arteriovenous malformations. One case, a patient with seizures and a family history of seizures, exhibited dysmorphic neurons and balloon cells, consistent with designation as FCD type IIb. Given the presence of FCD in proximity to an AVM, each of these cases can be classified as FCD IIIC. The presence of a cortical AVM might be predicted to lead to dysplastic cortical development. Literature characterizing the frequency of FCD in proximity to AVM is limited. Two large series suggest that the phenomenon is common and perhaps overlooked or not appreciated due on extent of surgical resection.

Conclusions

Both AVMs and FCD can provide a potential anatomic substrate for seizures, making it challenging to designate which abnormality was seizurogenic. In the case of FCD Type IIb with a family history of seizures, FCD was more likely to have been clinically relevant to her seizures.

11
Changes in FGF and WNT signaling in Arx mutant mice lead to defects in cortical functional domain development

Background

The mammalian cerebral neocortex is comprised of a 6 layer sheet with conserved functional domains distributed anterior to posterior and medial to lateral. Coordinated development of these cortical areas, along with the accompanying neural circuits, are essential for normal cognition, emotional responses, motor activity, and social behaviors of the brain. How cortical arealization is established from the naïve neural precursor cells in the early neural tube to the mature brain remains incompletely understood.

Methods

We have established that the aristaless-related homeobox (Arx) gene participates in patterning by suppressing ventral identity during cortical development. To further delineate the role of Arx in maintaining proper levels of morphogen expression, cortical arealization, and cortical circuit formation, we interrogated conditional mutant mice and the WNT and FGF signaling pathways.

Results

Our data show that the loss of Arx leads to disruption in FGF and WNT signals, which in turn alters positional identity within the neural precursors in the telencephalon as well as that of the mature neurons in the neocortex (i.e., shifts cortical area boundaries). These changes also results in defects in cortical circuit formation.

Conclusions

Together our data provide insight into how mutations in ARX cause abnormal brain development that leads to functional defects in human.

12
Clinical and neuropathologic findings in a child with NAXE gene variant

Background

The NAD(P)HX epimerase (NAXE) gene, formerly known as apolipoprotein A-I-binding protein (APOAIBP), encodes an enzyme in the NAD(P)H repair system which prevents accumulation of toxic metabolites. Pathogenic variants in this gene have been reported in progressive encephalopathy and/or neurometabolic diseases. A recent study has found that variants in NAXE are also associated with Leigh syndrome. Leigh syndrome, or subacute necrotizing encephalomyelopathy, is a progressive neurodegenerative disorder primarily presenting in infancy or early childhood. It is neuropathologically characterized by necrotic lesions with vacuolization, gliosis and vascular proliferation, with relative preservation of neurons.

Methods

A 3-year-old female with severe neurodevelopmental delay, autonomic instability, presumed syndrome of inappropriate antidiuretic hormone with profound hyponatremia, and recurrent urinary tract infections due to vesicoureteral reflux, was subsequently found to have a hom*ozygous c.206A>G (p.Asp69Gly) variant in the NAXE gene. She was reportedly normal until 9 months of age when she was diagnosed with a brainstem infarct and became ventilator dependent. A full autopsy was performed after she died from sepsis.

Results

Postmortem neuropathologic examination revealed bilateral cystic and gliotic encephalomalacia with spongy change, diffusely involving the neocortex and brainstem. There was prominent capillary proliferation, with relatively preserved neurons adjacent to the hypervascularity. Patchy, perivascular lymphohistiocytic infiltrate and microglial activation were also present. The pons showed foreign body giant cell reaction surrounding cholesterol clefts and abundant Rosenthal fibers.

Conclusions

Leigh syndrome is caused by mutations in either the mitochondrial or nuclear genome and encompasses a wide clinical and genetic spectrum. This is the first report to link a NAXE variant to Leigh syndrome-like histopathologic findings on an autopsy study. Since clinical and neuropathologic manifestations of this variant are unknown, it is unclear whether these changes can be attributed solely to the pathogenic role of the NAXE variant or sequelae of infarct, possibly reflecting combined effects of both.

13
Neuropathologic Features of Pallister-Killian Syndrome: Case Report and Literature Review

Background

Pallister-Killian syndrome (PKS) is a rare and severe disorder defined by mosaic tetrasomy of 12p. The disease is characterized by dysmorphic features, central nervous system malformations, and cardiac anomalies. The prognosis for PKS is poor.

Methods

A consented autopsy and neuropathologic examination was performed. A literature review for pathologic manifestations of PKS was performed.

Results

Literature review revealed numerous neuroimaging reports, limited autopsies (less than 10), and no dedicated neuropathology reports of patient with PKS. The infant male was born at 27 weeks gestation. PKS was verified by antemortem genetic testing which showed mosaic tetrasomy of 12p. Medical conditions included bronchopulmonary dysplasia, pulmonary hypertension, cardiac defects, and bacterial pneumonia. Neuroimaging revealed progressive cortical and white matter loss, and subdural and intraventricular hemorrhage. He died at 10 months of age. Autopsy revealed dysmorphic features including frontal bossing, high arched cleft-palate, depressed nose, low set ears, and webbed neck. Body measurements demonstrated increased head circumference, short body length, and shortened upper extremities. Neuropathologic examination revealed a low brain weight for age (757 grams), bilateral thin subdural hematomas, polymicrogyria, and globally diminished white matter. The brainstem and spinal cord were grossly normal. Sections showed abnormal cortical development including neuronal marginal heterotopia embedded in interhemispheric leptomeningeal bands, and focal cortical laminar disorganization. Amphophilic globules and dystrophic mineralization were present throughout the cerebrum and spinal cord, highlighted by Von-Kossa stain. Other findings included globoid cardiomegaly, right ventricular hypertrophy, atrial and ventricular septal defects, pulmonary artery dilation, bronchopulmonary dysplasia, absent left adrenal, and atrophic right testicl*.

Conclusions

PKS can present with a spectrum of central nervous system findings including structural abnormalities, malformations of cortical development, and white matter disease. In this case, leptomeningeal heterotopia, polymicrogyria, and cortical laminar disorganization suggest disrupted neuronal migration and cortical development. Further autopsy contributions are essential for elucidating PKS’s definitive gross and microscopic manifestations.

14
Cortical, white matter, and dentate gyrus atrophy associated with infantile myasthenic/hypotonia syndrome and heterozygous mutations in ALG14

Background

A 4-month-old girl born at term with congenital myasthenic/hypotonia syndrome, infantile spasms and heterozygous mutations (c.220g>A pAsp74Asn; c.203A>G p.Tyr68Cys) in the asparagine-linked glycosylation 14 (ALG14) gene presented for worsening seizure activity, aspiration, emesis and nocturnal hypoxemia. Unfortunately, epilepsy interventions were unsuccessful and the patient was transitioned to comfort care.

Methods

The family requested a full autopsy and donated the patient’s right hemisphere and muscle to ongoing congenital disorders of glycosylation (CDG) research at the NIH to contribute better understanding of this rare CDG.

Results

At autopsy, acute bilateral pneumonia was present. The frontoparietal lobes and corpus callosum showed marked atrophy associated with ventriculomegaly. Microscopically, acute and subacute white matter infarcts were present. Moderate loss of granular neurons were present in the dentate gyrus and there was marked cerebellar dentate nucleus injury out of proportion to the concurrent mild global acute hypoxic-ischemic encephalopathy. NIH results are not yet available.

Conclusions

CDGs are multisystem disorders with varying CNS functionality and neurologic symptoms. The ALG14 gene encodes a subunit of UDP-N-acetylglucosamine transferase, an enzyme which adds the second N-acetylglucosamine residue during synthesis of the lipid linked oligosaccharide in the endoplasmic reticulum. Autopsies of ALG14-CDG patients showed an early severe neurodegeneration with myopathic and myasthenic features. Two of these patients showed frontoparietal atrophy and clinical epilepsy. Importantly, one of these patients shared the same heterozygous mutation as this patient. Other patients in the series showed either delayed white matter myelination or subsequent white matter volume loss and ventriculomegaly. While hippocampal dentate gyrus injury was not described in this series, it is well known to be associated with hypoglycemia, which may be present in CDGs. Abnormal metabolic derangement may also be the underlying cause of the cerebellar dentate nucleus injury. Together, these findings strengthen and extend our knowledge of expected abnormalities in rare CDGs with ALG14 mutations.

15
Congenital smooth muscle hamartoma overlying a fibrous stalk: A potential cutaneous marker of occult spinal dysraphism

Background

Spinal dysraphism results from incomplete midline closure during neurulation. Occult spinal dysraphisms (OSDs) often manifest with overlying cutaneous stigmata. Fibrous stalks, a type of OSD, are frequently associated with” cigarette burn” lesions, sacral dimples, lipomas, and cutaneous vascular lesions, including infantile hemangiomas and capillary vascular malformations. We present a case of a fibrous stalk associated with an overlying congenital smooth muscle hamartoma (CSMH).

Methods

A male infant prenatally diagnosed with T11 hemivertebra and rib abnormalities was born at full term with dextroscoliosis, a closed-base sacral dimple, and an erythematous lesion over the lower thoracic spine. Postnatal imaging revealed spinal dysraphism with a subcutaneous fibrous sinus tract and tethered low-lying conus medullaris. At 16-months of age, spinal cord detethering was performed. Intraoperatively, a tract extended from the thoracic cutaneous lesion through the soft tissue and dura and attached to the spinal cord. The intra- and extradural tract, including the skin, was sent en bloc for pathologic examination.

Results

Histologic sections revealed a disorganized proliferation of smooth muscle within the reticular dermis and superficial subcutis, consistent with CSMH. A cord-like stalk of benign fibrous tissue with nerve twigs, meningothelial cells, and focal cartilage extended from the CSMH into the soft tissue. There was no evidence of an epithelium-lined sinus tract, neuroglial tissue, or vascular malformation.

Conclusions

CSMH is an uncommon benign cutaneous lesion composed of a proliferation of smooth muscle thought to arise from arrector pili muscle. Clinically they can be confused with other congenital cutaneous lesions including vascular malformations. CSMHs have not been previously associated with OSDs. We present the first reported case of a CSMH overlying a fibrous stalk. This case illustrates a previously unrecognized cutaneous lesion that could potentially herald an underlying OSD.

16
Cortical dyslamination and dysmorphic neurons in POLG-related mitochondrial disorder

Background

Biallelic variants in the nuclear POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase γ, are the most common cause of inherited mitochondrial disorders and result in a wide phenotypic spectrum of diseases including progressive external ophthalmoplegia and sensory ataxic neuropathy, with age of onset ranging from infants to adults.

Methods

We report brain and muscle biopsy findings from a patient with POLG-related disorder with the following POLG variants: maternal allele: c.2243G>C (p.W748S); paternal allele: c.1399G>A (p.A467T). A 29-year-old pregnant female with recent history of mild COVID-19 presented with refractory status epilepticus. Magnetic resonance imaging (MRI) of the brain showed waxing and waning, diffusion restricting T2/FLAIR hyperintense lesions in the periventricular white matter, frontal, parietal, and occipital lobes, and posterior cingulate gyrus as well as mild diffuse pachymeningeal enhancement. Extensive laboratory workup was negative for infectious or autoimmune etiologies. Three separate biopsies/resections of the MRI lesions in the parietal and frontal lobes and a muscle biopsy of the thigh were performed.

Results

The brain showed severe patchy astrogliosis and microglial activation with rare microglial nodules and sparse perivascular lymphocytic infiltrate. Additionally, disorganized cortical lamination with scattered dysmorphic neurons resembling focal cortical dysplasia (FCD) International League Against Epilepsy (ILAE) type 2A was seen. On electron microscopy studies of the parietal lobe, there were rare filiform mitochondria. The muscle demonstrated critical illness myopathy with no COX negative fibers. Ultrastructural studies revealed large areas of myofibrillar abnormality consisting of loss of the A-band, I-band and M-line with some mitochondria showing filiform changes.

Conclusions

Cortical dysplasia can be seen in mitochondrial diseases but has not been reported previously in POLG-related disorder. Although coincidental FCD cannot be excluded, the patient had no history of seizures prior to the current presentation. Our findings expand the neuropathologic spectrum of POLG-related mitochondrial disorder.

17
Hyaline Protoplasmic Astrocytopathy in Two Surgical Cases of Developmental and Epileptic Encephalopathy

Background

Hyaline protoplasmic astrocytopathy (HPA) is a rare condition characterized by astrocytic cytoplasmic inclusions, distinct from Rosenthal fibers, in the cerebral cortex of unknown pathogenesis and clinicopathological significance. We performed immunohistochemical and electron microscopic studies and proteomic analysis of HPA inclusions using FFPE surgical specimens from two patients with developmental and epileptic encephalopathy.

Methods

Case 1 is a 2-year-8-month-old boy presenting with tonic seizures and epileptic spasms since 3 months old, and was diagnosed with West syndrome followed by Lennox-Gastaut syndrome. Brain MRI revealed polymicrogyria in the right frontal lobe. ACTH therapy and total corpus callosotomy (TCC) were ineffective. He became seizure-free after multistage right hemispheric disconnective surgeries. Case 2 is a 10-year-1-month-old boy with Lennox-Gastaut syndrome. He developed epileptic spasms at 7 months old, which resolved temporally with ACTH and other therapies until 5 years old. MRI revealed abnormal gyral formation in the right frontal lobe. The patient became seizure-free after TCC and subsequent right frontal lobe disconnection.

Results

Histopathological examination of the frontal lobe biopsy specimen revealed polymicrogyria with HPA in both cases. Inclusions were brightly acidophilic, PTAH-positive, and non-amyloid, and variably immunoreactive for S-100, glutamate transporter-1, aldehyde dehydrogenase 1 family member 1, cytoglobin, and HSP70; no immunoreactivities were observed for GFAP, filamin A, ubiquitin, p62, or alpha B-crystallin. GFAP expression in most cortical astrocytes was faint or absent. Electron microscopy revealed inclusions consisting of densely packed electron-dense granules of approximately 30-40 nm diameter without unit membranes or filamentous structures, intermingled with occasional mitochondria and Golgi apparatus. Proteomic analysis of macrodissected cerebral cortex by quantitative mass spectrometry detected significantly upregulated astrocytic proteins, including glutamine synthetase and tenascin C, in HPA compared to control cases.

Conclusions

These results suggest that HPA inclusions are derived from organelles and that HPA represents an altered astrocytic glutamate metabolism secondary to intrauterine encephaloclastic mechanisms and neuronal hyperexcitation.

18
SEQUENTIAL EMERGENCE AND CONTRACTION OF EPITHELIAL SUBTYPES IN THE PRENATAL HUMAN CHOROID PLEXUS REVEALED BY A STEM CELL MODEL

Background

As the blood-cerebrospinal fluid interface, the choroid plexus (ChP) mediates body-brain communication throughout life and has broad potential for CNS regenerative medicine. Despite this, relatively little is known about the human ChP or its epithelial cells (CPECs), including their lineages.

Methods

Based on an earlier proof-of-concept method, we devised a simple, efficient, and scalable protocol for CPEC derivation from human pluripotent stem cells. Time-series analysis of single-cell RNA-sequencing data were then performed and validated in independent derivations and perinatal tissues.

Results

In the absence of mesenchymal elements, derived CPECs (dCPECs) acquired canonical properties and displayed dynamic multiciliated phenotypes that impacted Aβ uptake and paralleled those seen in human tissues. Single dCPEC transcriptomes correlated well with human organoid and fetal CPECs, while pseudotemporal and cell cycle analyses revealed dCPEC, neuron, and neural progenitor lineages arising from neuroepithelial cells. In addition, transcriptome analyses defined metabolic (type 1) and ciliogenic dCPECs (type 2) at early stages, followed by type 1 diversification into anabolic-secretory (type 1a) and catabolic-absorptive subtypes (type 1b) as type 2 cells contracted. The sequential emergence and contraction of these subtypes was then confirmed in independent derivations and mapped to distinct prenatal stages using human tissues.

Conclusions

These findings establish an improved protocol for deriving human CPECs, define their prenatal lineage dynamics, and lead to new models of ChP/CSF functions during human brain development.

19
Neuropathologic findings in a 32-year-old man with Phelan-McDermid syndrome

Background

Phelan-McDermid syndrome (PMS), also known as 22q13.3 deletion syndrome, is a rare genetic disorder typically involving the loss of the SHANK3 gene affecting neurological development and leading to facial abnormalities and intellectual disabilities, including features of autism spectrum disorder. Neurofibromatosis type II (NF2) is caused by mutations in the nearby NF2 gene (22q12.2), with well-documented neuropathologic features. In contrast, the tissue neuropathology of individuals with PMS has not been reported.

Methods

We contribute the complete neuropathology of an autopsy case of a 32-year-old man with a diagnosis of Phelan-McDermid syndrome confirmed on antemortem detection of chromosome 22q13 deletion (further details unavailable).

Results

The brain was micrencephalic (weight, 900g; posterior fossa contents, 90g), with slight cerebral and cerebellar atrophy, and meningiomas in the parietal dura (0.3cm) and the glomus of the choroid plexus (0.7cm). The hippocampi were asymmetric, the left having a globular configuration, with subicular widening and expansion of the temporal horn of the lateral ventricle. Microscopic examination further revealed a 0.1cm glial hamartoma in the amygdala, a focus of meningothelial hyperplasia in the basal leptomeninges, and a Schwann cell tumorlet (0.1cm) in the trigeminal nerve. A clear concurrent diagnosis of NF2 by conventional Manchester (NIH) criteria could not be made, however.

Conclusions

Given the proximity of the causative mutations for PMS and NF2 on chromosome 22, we suggest that this decedent may have had a heretofore unrecognized “overlap” syndrome. Investigators have hypothesized that patients with both PMS and NF2 may have a ring chromosome 22 (forming after the loss of genetic material from both the short and long arms), leading to instability of the chromosome during mitosis. Whether other patients with PMS (with or without ring chromosome) also have neuropathologic features of NF2 will require additional autopsy evaluation.

20
Autopsy findings in a patient with a severe neurodevelopmental disorder due to compound heterozygous mutations in PNPT1

Background

Heterozygous mutations in PNPT1 gene (which encodes polynucleotide phosphorylase, a mitochondrial 3’-to-5’ exoribonuclease involved in mtRNA processing) cause autosomal dominant spinocerebellar ataxia 25, while patients with bi-allelic PNPT1 mutations exhibit wide clinical heterogeneity, ranging from non-syndromic hearing loss to multi-system Leigh disease-like syndrome. The underlying pathologic changes have not been reported and disease pathogenesis is poorly understood, with mitochondrial dysfunction and systemic inflammation (due to activation of interferon type I response by accumulated double-stranded mtRNA) proposed as possible disease mechanisms.

Methods

Here, we report the autopsy findings from an 11-year-old boy with compound heterozygous PNPT1 mutations, clinically manifesting as cerebral palsy, intellectual disability, dystonia, epilepsy, vision and hearing impairments, scoliosis, dysphagia, constipation, and cyclic vomiting.

Results

At autopsy, the CNS showed severe microcephaly due to cerebral leukoencephalopathy, with widespread white matter attenuation and severe thinning of corpus callosum; pontocerebellar degeneration, with axon loss in the middle cerebellar peduncles and base of pons, patchy loss of Purkinje and dentate nucleus neurons, cerebellar white matter degeneration, and severe atrophy of the cerebellar vermis; and spinocerebellar atrophy, with degeneration of dorsal spinal columns and afferent cerebellar tracts. Cranial and peripheral nerves showed moderate-severe axonal polyradiculoneuropathy with focal demyelinating features, with greater involvement of sensory than motor cranial nerves and dorsal than ventral spinal roots. Skeletal muscles showed ultrastructural mitochondrial abnormalities and chronic neurogenic changes accompanied by ongoing denervation. The gastrointestinal system demonstrated widespread loss of interstitial cells of Cajal and patchy thinning of the colon wall due to smooth muscle degeneration and fibrosis. Superimposed acute ischemic changes were seen in multiple organ systems. Aside from focal bronchopneumonia, no significant inflammation was seen.

Conclusions

Overall, these autopsy findings are consistent with a systemic mitochondrial disorder and do not provide evidence for a concurrent neuroinflammatory disease process, providing important insight into the pathogenesis of this rare disease.

21
Artificial Intelligence-Based Statistical Modeling Delineates Cortical Architectural Distortion in Surgically Resected Epileptogenic Foci

Background

In epilepsy patients lacking radiographic evidence of focal cortical dysplasia (FCD) or other epileptogenic lesions, surgical resections target regions of abnormal electroencephalographic activity. Identifying cortical architectural disorganization in these specimens by conventional histology remains challenging. We explored artificial intelligence (AI)-based cell mapping and morphometry combined with spatial statistical models to evaluate neuronal architectural distortion within and adjacent to regions of FCD.

Methods

Sections from FCD resections (n = 29) and matched regions from neurologically healthy autopsy controls (n = 19) were stained with NeuN. Whole slide images were generated, and regions of interest (ROI) were annotated in QuPath for surgical samples as FCD (n = 33), FCD-adjacent (n = 14), Apparently-normal (n = 36), and Autopsy True-normal (n = 19). The QuPath cell detection algorithm was used to identify NeuN-positive cortical neurons and compared to manual annotations. Extracted spatial and morphometric data for each cell was analyzed in R 4.2.1 using SpatStat. A multinomial, LASSO-regularized regression classifier was trained and tested on an 80/20% training/test split. Kruskal-Wallace tests with non-parametric post-hoc comparisons were used for cell morphologic features, and permutations tests were used to compare summary functions of neuronal spatial distribution.

Results

The cell detection algorithm had an overall sensitivity of 90% and a false positive rate of 12%, with no significant difference in accuracy across categories. The multinomial classifier yielded an accuracy of 70.5 and 82.5% for the ROI classification and overall specimen diagnosis. Parameters related to neuronal clustering, inhom*ogeneity of neuronal distribution, and nuclear size and regularity factored prominently into the classifier. Subsequent statistical analyses demonstrated significant differences in the intensity of neuronal point patterns (p = 0.01), the degree of spatial inhom*ogeneity (p < 0.001), and degree and distribution of neuronal clustering (p < 0.001) across the annotated categories.

Conclusions

AI-based quantification of cellular morphology and spatial distribution combined with spatial statistical modeling may augment the detection of abnormal neuronal architecture in surgically-resected epilepsy foci.

22
A Rare Case Involving Fetal Immunodysregulation Polyendocrinopathy Enteropathy X-linked (IPEX) Syndrome

Background

This case involved a male fetus that was the product of a 26-week gestation to a 25-year-old primigravid woman. The pregnancy was complicated by multiple anomalies seen on prenatal ultrasound including fetal hydrops. The mother initially presented with severe back pain consistent with contractions and underwent spontaneous vagin*l delivery of the fetus. A fetal hydrops genetic panel was pursued, which showed a hemizygous mutation for a likely pathogenic variant in FOXP3 (c.17del, p.Pro6Leufs*56) and heterozygous mutation for a variant of uncertain significance in RPS24 (c.734C>T, p.Pro245Leu).

Methods

At neuroautopsy, the fresh brain weighed 79.2 grams (normal range for 26-weeks gestational age is 105.0 +/- 19.0 grams). External examination of the brain showed a diffuse dusky red-brown discoloration noted throughout the leptomeninges. Coronal sections of the cerebral and cerebellar hemispheres showed moderate-to-severe maceration.

Results

Neuropathologic examination of the tissue sections demonstrated multifocal areas of extensive fresh hemorrhage, transcortical neuronal loss, laminar necrosis with gliosis, small vessel proliferation, and sheets of macrophages. Interestingly, there was a prominent lymphocytic population of infiltrates associated with the areas of organizing cortical necrosis.

Conclusions

This fetus shows a rare presentation of immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome. IPEX syndrome is a rare disorder resulting in aggressive autoimmunity and early death and is due to a mutation in FOXP3 which results in dysfunctional T-regulatory cells. Most patients die in infancy or childhood, though some patients have lived longer with chronic immunosuppression or bone marrow transplants. Patients usually present with infantile diabetes, severe enteropathy, eczema, anemia, and hypothyroidism. Histologic findings are striking, and lymphocytic inflammatory infiltrates of multiple organs. IPEX is an X-linked disease, resulting in female carriers and males with disease. In some families the affected males present earlier and much more severely. In these families the most common presentation is familial intrauterine demise of male fetuses in midtrimester.

23
Characterization of a Population of Neural Progenitor Cells in the Developing Hippocampus

Background

Hippocampal circuitry is critically involved in navigation and episodic memory. The development of the circuit is a finely regulated process which includes the approximation and recession of the lateral ventricle ependyma. As the ependyma retreats, it forms a “seam” below Ammon’s horn, which we refer to as the subammonic seam. In this process a population of candidate neural progenitor cells is left hundreds of microns from the ventricle, effectively ‘migrating’ due to the seam closure. Our study aims to characterize this population of cells.

Methods

Autopsy cases for spontaneous fetal demise or infant death between 20 and 144 weeks post conception were identified. Approximation of the ependyma, seam closure, and position of candidate progenitor cells were assessed. We are evaluating morphologic and immunostaining findings to visualize seam closure and candidate progenitor cell persistence, movement, morphological change, and protein expression.

Results

The subammonic seam begins to close in the third trimester. Candidate neural progenitor cells could be morphologically identified along the closing seam out to 56 weeks post-conception. The subammonic seam can be definitely identified for months after birth until it begins to recede in prominence then vanishes into the white matter of the mature parahippocampal gyrus.

Conclusions

The candidate neural progenitor cells persist along the seam for up to 16 weeks postnatally. This cell population could be implicated in postnatal neurogenesis after the recession of the germinal matrix within this region. Preliminary results suggest some possible trajectories. Further understanding of this cell population may provide a more complete picture of the formation of hippocampal circuitry and potentially of the pathogenesis of diseases such as epilepsy.

24
Hyaline Protoplasmic Astrocytopathy of the Neocortex: A Case Study and Literature Overview

Background

Hyaline Protoplasmic Astrocytopathy (HPA) is a rare disorder of uncertain etiology characterized by the presence of eosinophilic hyaline inclusions within the cytoplasm of protoplasmic astrocytes.

Methods

We examine histology and electron microscopy in brain resection tissue from a patient with chronic epilepsy and HPA, integrating case-specific findings with existing literature.

Results

A 10-week-old girl developed recurrent episodes of head drop associated with arm and leg extension. MRI showed diffuse microgyria and partial agenesis of the corpus callosum. Over subsequent years, she exhibited severe developmental delay, was non-verbal, and G-tube dependent. Extensive genetic and metabolic testing were unrevealing. EEG showed excess background theta slowing, brief bursts of generalized polymorphic delta activity, and abundant independent multifocal epileptiform discharges. Multiple antiepileptics were trialed, and at age 14 she underwent right midfrontal gyrus resection with bilateral centromedian/pulvinar thalamic nuclei responsive neurostimulation placement, all without improvement. Histologic sections showed cerebral cortex and subcortical white matter with numerous glial cells with optically clear nuclei containing abundant glassy, eosinophilic cytoplasmic inclusions present throughout the cortex. The inclusion material was negative for GFAP and weakly immunoreactive for S100. NeuN immunostaining showed focal neuronal depletion and tangential disarray with absence of distinct layering. Electron microscopy of the inclusions demonstrated relatively uniform, non membrane-bound, electron-dense granular material with areas of clearing containing lighter material possibly representing degenerating organelle elements. A diagnosis of Hyaline Protoplasmic Astrocytopathy of Neocortex with Focal Cortical Dysplasia (ILAE type Ib) was rendered.

Conclusions

HPA is a non-specific constellation of findings associated with chronic seizure disorders in various settings, including Aicardi syndrome, tuberous sclerosis, and focal cortical dysplasia. The inclusions stain positive for Filamin A, a regulator of neuronal migration during development, and the glutaminate transporter GLT-1. As a very rare entity, further descriptions of patients with this entity will contribute to a better understanding of the pathophysiology.

25
Post-Treatment Neuropathologic Findings in Aicardi-Goutières Syndrome

Background

Aicardi-Goutières syndrome (AGS) is a rare inherited neurodegenerative disorder associated with interferon overproduction. The syndrome is characterized by inflammatory encephalopathy and cerebral calcifications. Janus Kinase (JAK) inhibitors, such as baricitinib and ruxolitinib, have been explored in the treatment of AGS, however, little is known about the neuropathologic impacts of JAK inhibitor therapy in AGS.

Methods

A consented, unlimited autopsy with formal neuropathologic examination of the brain and spinal cord was performed.

Results

The decedent was a 4-year-old boy with AGS diagnosed in the immediate postnatal period. Genetic testing at two months of age revealed a compound heterozygous mutation in RNASEH2C. He was subsequently initiated on baricitinib. At 4 years of age, he developed septic shock and acute respiratory distress syndrome as a result of aspiration pneumonia. Following compassionate withdrawal of care, he underwent a complete consented autopsy. Externally, body weight was below the 1st percentile for age. Dysmorphic features included microcephaly, equinovalgus feet, and microphthalmia. Internal examination revealed multifocal pneumonia and acute tubular injury. Neuropathologic examination revealed a 219.6-gram brain exhibiting markedly diminished white matter and extensive calcific deposits involving the cerebral cortex, white matter, deep gray nuclei, cerebellum, and brainstem. Microscopic examination revealed astrocytic gliosis and numerous, variably sized calcifications. There were perivascular, intraparenchymal, and leptomeningeal chronic inflammatory infiltrates, composed of CD4 positive T-lymphocytes, rare CD8 positive T-lymphocytes, and rare CD20 positive B-lymphocytes. CD68-positive macrophages were predominately perivascular. Luxol fast blue showed demyelination of the corpus callosum and subcortical white matter. Microinfarction and Bergmann gliosis were identified in the cerebellum. The spinal cord showed diminished bulk of the anterior horn. Calcifications did not involve the spinal column.

Conclusions

Neuropathologic findings in this case of AGS, post-JAK inhibitor therapy, underscore persistent neuroinflammation and degeneration. Further studies are essential to clarify the implications of these results and guide future therapeutic strategies.

26
Toxic/Metabolic Leukoencephalopathy in Children

Background

Toxic-metabolic leukoencephalopathy, characterized by significant white matter damage as the primary finding, is uncommon in children without prior significant neurologic history. We encountered two cases recently with history of a viral prodrome, new-onset seizures quickly progressing to status epilepticus and death, with autopsy findings of non-infectious leukoencephalopathy. On delving into the clinical history, the findings were most consistent with drug toxicity. We decided to review the autopsy cases at our institution over the past 10 years for a diagnosis of leukoencephalopathy/ leukodystrophy and correlated these findings with the clinical history.

Methods

Cases with predominant white matter injury/ myelin loss were identified from the autopsy database maintained by our neuropathologist, and cases with only perinatal hypoxic-ischemic injury were excluded. Complete autopsy reports for cases selected were reviewed.

Results

Seven cases with primary leukoencephalopathy/ leukodystrophy were identified out of 315 cases, with age at autopsy ranging from 6 days to 16 years. 2 had known metabolic disorders, and 2 had findings consistent with drug toxicity. Genetic mutations for metabolic disorders were identified postmortem for 2 cases and the findings on 1 case were suggestive of peroxisomal biogenesis disorder. All the cases had varying severity of associated hypoxic-ischemic injury, and some of them also had abnormal neuronal migration.

Conclusions

Presence of significant white matter damage at autopsy should raise the suspicion for toxic-metabolic disorders. In-depth review of clinical history can offer a clue to the diagnosis of drug- associated toxic leukoencephalopathy. Findings of toxic-metabolic leukoencephalopathy/leukodystrophy at autopsy are still very rare.

27
Spectrum of Meningoencephaloceles Presentation: From Massive to Atretic - Case Reports and Literature Review

Background

Meningoencephaloceles are neural tube defects that affect approximately 0.8-4 per 10,000 live births. They represent congenital malformations with protrusion of central nervous system structures through a cranial defect with associated arachnoid, cerebrospinal fluid, and dura. We report a case of a male infant with a prenatally diagnosed large occipital meningoencephalocele and a female infant with an occult presentation.

Methods

Clinical features were summarized from the patient’s medical record. A literature search was undertaken using relevant key words.

Results

Case A: A large meningoencephalocele was identified in utero in a male fetus with elevated maternal alpha-fetoprotein levels. Following Caesarean section delivery, imaging revealed an occipital encephalocele, 10.5 x 9.4 x 4.5 cm, associated with a midline defect in the posterior calvarium. Resection was undertaken and macroscopic examination identified nodules of malformed cerebrum enclosed by cystically dilated meninges associated with skin. Microscopic evaluation identified neuroparenchyma with dystropic lamination and calcifications. No evidence of cerebellar parenchyma or other hindbrain elements was identified. Case B: A 7-year-old girl presented with a midline posterior parietal scalp mass at birth. During early childhood, the lesion would increase in size while crying and was noted to be pulsatile. Imaging revealed normal intra-axial structures with persistent falcine and emissary veins extending to what was interpreted as an extracranial venous malformation in the parasagittal parietal scalp. Radiologic diagnosis was sinus pericranii and treatment was deferred. Tenderness and increased size, to 1.7 x 2.2 x 1.7 cm, lead to resection. Histologic evaluation revealed a minute nodule of dysplastic neuroparenchyma surrounded by arachnoid, dura, and skin.

Conclusions

The prognosis for occipital and parietal encephaloceles is related to the size and location of the encephalocele, the presence or absence of cerebellum or other hindbrain contents within the herniated tissue, the occurrence of hydrocephalus, and the presence or absence of concurrent cerebral malformations.

Posters: Infectious
28
A case of racemose neurocysticercosis with molecular diagnosis

Background

A 40-year-old, otherwise healthy, Spanish-speaking male immigrant from Mexico presented to the Emergency Department with one and a half months of progressive speech difficulty, headaches, and confusion. Neurologic examination was notable for expressive aphasia with intact facial and tongue movement. Computed tomography with angiography demonstrated a massive loculated cystic mass in the left frontotemporoparietal lobes measuring 13.6 x 6.6 cm and causing midline shift and brainstem compression. Magnetic resonance imaging showed that the lesion was non-enhancing except along septa and had a similar intensity to cerebrospinal fluid. The patient subsequently underwent decompressive hemicraniectomy due to acute worsening of his neurologic state.

Methods

Macroscopic evaluation showed a fragmented, tan-pink lesion with both cystic and solid components. H&E-stained sections demonstrated a collapsed, redundant cyst wall with three layers: a brightly eosinophilic cuticle layer reminiscent of broad, blunt villous structures; a more cellular "pseudoepithelial" layer composed of non-human cells; and a loose, reticular layer. Small amounts of reactive brain tissue, foreign body giant cells, and rare calcification were present. No definitive evidence of a parasite, such as hooklets, scolex, or spinal canal, was identified.

Results

Laboratory workup was positive for cysticercosis IgG and echinococcus IgG antibodies. The two IgG’s are however known to cross-react, warranting molecular testing. PCR testing on the surgical specimen was positive for Taenia solium, confirming a diagnosis of neurocysticercosis. He was treated with albendazole and praziquantel and had returned to baseline function without recurrence at follow-up of one year.

Conclusions

Neurocysticercosis is the commonest invasive parasitic infestation of the central nervous system but is an infrequent surgical specimen in the United States. Racemose neurocysticercosis is a relatively rare form of neurocysticercosis. Racemose neurocysticercosis forms abundant grape-like clusters of cysts but often lacks a scolex. In this case, molecular testing was used to confirm the infectious organism.

29
Intracranial Cerebral Mucormycosis Infection in the Context of Immunosuppression: Comparison of Ischemic vs. Hemorrhagic Complications

Background

Fungal infection of the central nervous system (CNS) is rare and most commonly affects immunocompromised patients. We report two cases of CNS mucormycosis, presenting variably with infarct or hemorrhage.

Methods

Clinical features were summarized from the patients’ medical records. Key histopathologic features were reviewed. A literature search was undertaken using relevant key words.

Results

Patient A: A 75-year-old man with a history of chronic lymphocytic leukemia, undergoing targeted therapy with Imbruvica (ibrutinib), presented with headache, decline in cognitive ability, generalized weakness, and blurry peripheral vision in the right eye. CT and MRI scans identified right maxillary sinus air-fluid levels and left parietoccipital region abnormalities raising the differential diagnosis of a subacute infarct and a high-grade neoplasm and resection was undertaken. Case B: A 23-year-old man with a history of diabetes mellitus type 1 presented with diabetic ketoacidosis. Chest CT scans identified multiple areas of consolidation in his lungs. Initial CT scan of his brain was negative with opacification of the right sphenoid sinus. After being found unresponsive repeat CT scan identified a right frontal lobe parenchymal hematoma which was evacuated. In both cases histologic evaluation revealed angioinvasion by broad, pauciseptate hyphae most consistent with mucormycosis with associated infarct in Case A and hemorrhage in the context of a ruptured, infected artery in Case B. Infection was not in the clinical differential and culture was not performed for either case. While ischemic injury is most commonly identified in the setting of mycotic vasculitis, occasional cases manifesting with hemorrhage have been reported.

Conclusions

This report compares and contrasts two cases of central nervous system mucormycosis that resulted in different cerebrovascular complications, in order to compare precipitating factors and suggest why some patients may be predisposed to aneurysm or intracranial hemorrhage, resulting in progressive disease and poor prognosis.

30
HTLV-1 Associated Myelopathy (HAM): Case Report and Literature Review

Background

HTLV-1 infection is endemic in many areas of the world but there is a low prevalence in the US (about 1%). Here we present an 85-year-old Haitian female with a 15-year history of paraplegia secondary to HTLV-1-associated myelopathy (HAM), also known as tropical spastic paraparesis (TSP).

Methods

The patient presented to Beth Israel Deaconess Medical Center with altered mental status and respiratory failure. Medical imaging showed chronic intracerebral microvascular ischemic changes. Her hospital course was further complicated by sepsis. The patient quickly decompensated and died and a complete autopsy was performed.

Results

There was a macroscopic thinning of the solar and lumbar plexi. There was extensive loss of axons with an associated gliosis in the lateral and anterior columns of the spinal cord and patchy perivascular lymphocytic infiltrates microscopically. The damage extended from the cervical cord to the lumbar cord. There was preservation of the anterior horn neurons. Neurofilament protein immunohistochemistry demonstrated a symmetric loss of axons in the lateral columns of the spinal cord. Cortical sections demonstrated a mild thickening of the leptomeninges and a subpial gliosis.

Conclusions

HAM/TSP is a rare condition within the United States marked by meningomyelitis with axonal degeneration and associated gliosis predominately affecting the lateral columns of the thoracic cord. There can be variable damage to the anterior and posterior columns. In order to establish the diagnosis of HAM, other infectious, autoimmune, and neurodegenerative etiologies of spinal cord degeneration need to be excluded. Many of the infectious agents (i.e., polioviruses) causing transverse myelitis are neuronotrophic and damage the anterior horns. The autoimmune causes (i.e., multiple sclerosis and vasculitis) have variable patterns of distribution, but in general show axonal preservation. The neurodegenerative causes (i.e., amyotrophic lateral sclerosis with or without frontotemporal lobar degeneration) have a similar distribution but have an intracranial component in addition.

31
Polymicrobial brain abscesses: A clinicopathological study of 30 cases

Background

Brain abscesses (BAs) are focal parenchymal infections that are caused by a range of pathogens; despite advances in the diagnosis and treatment during the past decades, they remain a life-threatening condition. Polymicrobial BAs (PBAs) involve two or more infectious microorganisms within their foci; they are complex coinfections with diagnostic and therapeutic challenges.

Methods

We studied 30 patients (ages: 26 ∼ 78 years, with a mean of 58 years) with PBAs that were histopathologically examined in correlation with radiological and clinical features.

Results

These patients included 6 females and 24 males; 9 diabetic and 3 other immunocompromised individuals. Common clinical presentations included headache (12/30), confusion (11/30), fever (5/30), hemiparesis (5/30), decreased level of consciousness (5/30), and seizure (4/30). PBAs tended to occur more often in the left (20/30) than the right (10/30) cerebral hemisphere; the most common location was the frontal lobe (17/30), followed by the parietal lobe (6/30); PBAs were unifocal (29/30) except in one case with radiologically multifocal disease within the frontal lobe. While PBA imaging was mostly characterized by a ring-enhancing lesion/mass with central restricted diffusion, some cases showed complex imaging features such as increased heterogeneity, meningeal involvement (10/30) or associated ventriculitis (6/30). PBA histopathological features varied from encephalitis/cerebritis with or without necrosis (9/30, early stages) to encapsulated foci (21/30, late stages); some of which demonstrated complex changes with one pathogen predominating features in certain areas and another pathogen in other areas. The causative pathogens were exclusively bacteria in most cases (25/30); coinfections of bacteria with Toxoplasma (2/30), fungi (2/30) and virus (1/30) were also identified. Streptococcus species (16/30) were the most common pathogen.

Conclusions

Our findings suggest that compared to monomicrobial/overall BAs, PBAs share some similar features such as left-sided predominance, but they are usually unifocal and complex with increased heterogeneity on imaging and histopathology.

32
Chronic Meningoencephalitis Caused by Coxsackievirus B3 in a Patient Treated With Ocrevus® (Ocrelizumab) for Multiple Sclerosis: A Case Study

Background

Coxsackievirus B3 (CVB3) is a common human pathogen that predominantly causes a self-resolving mild flu-like illness. CVB3 neurotropism makes it the leading cause of aseptic meningoencephalitis in young children and a rare complication in patients on immunosuppressive medications, such as rituximab. Less is known about the potential infectious side effects of ocrelizumab, an anti-CD20 monoclonal antibody recently FDA-approved for the treatment of multiple sclerosis (MS).

Methods

We present a case of chronic meningoencephalitis caused by CVB3 in a 50-year-old woman with a 9-year history of MS undergoing ocrelizumab treatment for approximately 3 years. Despite stability of her MS condition, the patient presented with a subacute decline in mental status and cognition. Upon admission, imaging showed predominant bilateral frontal periventricular white matter lesions, and a lumbar puncture revealed elevated protein, normal glucose, 1-2 nucleated cells, no oligoclonal bands, and a negative infectious workup (including HSV, JC virus, CMV, toxoplasma, and bacterial cultures), suggesting a demyelinating etiology. A brain biopsy of these lesions revealed a generalized inflammatory process with lymphocytic infiltration of the meninges without clear evidence of demyelination. After minimal improvement over 3.5 weeks of intravenous steroids, the patient expired and underwent a full autopsy.

Results

Postmortem neuropathologic examination of the brain showed scattered microglial nodules with neuronophagia, multifocal neuronal depletion, and diffuse parenchymal and perivascular lymphocytic inflammatory infiltrates involving the cerebral cortex and midbrain. Further evaluation of CSF using metagenomic sequencing identified Coxsackievirus B3, and RNAScope probes for Coxsackievirus B3 highlighted viral genomic RNA in neuronal bodies and at the center of microglial nodules.

Conclusions

This case study highlights the importance of considering atypical infectious etiologies in patients with neuroinflammatory conditions, particularly in the setting of immunosuppression, and adds CVB3 meningoencephalitis as a rare and unanticipated morbidity associated with ocrelizumab.

33
CMV-HSV1 VENTRICULO-ENCEPHALITIS WITH HISTOLOGICALLY OCCULT HSV1

Background

A 24-year-old man had subacute onset of generalized weakness, confusion, bilateral arm paresthesia, and slurred speech.

Methods

Case report

Results

Head CT demonstrated a 4.4 cm in diameter hyperdense lesion with surrounding hypodensity in the left frontoparietal lobe, suggesting a hemorrhagic lesion with vasogenic edema. The patient was found to have generalized lymphadenopathy and pancytopenia with WBC 0.2 x103 cells/µL, Hgb 7.9 g/dL, and platelets 14 x 103/mL. Tested for HIV-1, he had a plasma viral load of 3 million copies/mL (log10 6.49 cp/mL) and a CD4 count of 159 cells/mm3. He was started on antiretroviral therapy. Worsening clinical status prompted a craniotomy with hematoma evacuation, and histology yielded a diagnosis of EBER-positive DLBCL. The patient received chemotherapy and whole-brain irradiation. The clinical course deteriorating the following six months by disseminated histoplasmosis, CMV viremia, hydrocephalus (due to leptomeningeal involvement), and neutropenia. The patient progressively worsened with continuous altered mental status and emergence of ganciclovir-resistant CMV viremia; he was made DNR in comfort care and expired. Brain autopsy revealed: no active lymphoma, leptomeningeal foci of hemorrhage, necrotizing ventriculo-encephalitis with prominent “owl eye” CMV inclusions, and sub-acute gray matter infarction. This latter with extensive selective neuronal necrosis in the absence of viral inclusions or cellular response. Positivity for CMV Immunostaining was seen in the necrotic regions associated with inclusions. Immunostaining for HSV1, but not CMV, was extensively positive in areas with selective neuronal necrosis, such as the hippocampus, characterizing a rare HSV1-related pseudoischemic change, as described in the literature.

Conclusions

We present a HIV/AIDS young patient with CNS lesions attributable to three Herpesviridae viruses: EBV, CMV and occult HSV1. The areas showing HSV1 immunostaining displayed selective neuronal necrosis without Cowdry type A inclusions. Pseudoischemia is an unusual manifestation of HSV that may not be widely appreciated.

34
A Case of Whipple Disease in the Brain

Background

Whipple disease is a rare systemic infectious disorder caused by bacterium Tropheryma whipplei. Whipple disease involving central nervous system is a very rare condition, and research on this specific aspect is limited.

Methods

We describe a case of a 69-year-old male who presented with an episode of confusion, weight loss, worsening memory, and right sided weakness over the last month. The patient was started on antibiotics to treat potential urinary tract infection. His further workup included brain MRI which revealed a 2.4 cm enhancing mass centered in the left lentiform nucleus, with extension to the left caudate nucleus.

Results

The patient underwent left insula lesion stereotactic biopsy, and the obtained specimen consisted of multiple small fragments of firm white granular tissue. Microscopy showed granulomatous inflammation with numerous large reactive astrocytes and lymphocytic infiltration with perivascular cuffing.  The biopsy tissue was sent for Broad-Range PCR and Next Generation Sequencing, where Tropheryma whipplei was detected. The patient was started on intravenous ceftriaxone. Several days later, the patient underwent duodenal and gastric biopsy, which revealed duodenal mucosa with preserved villous architecture and focal gastric metaplasia, suggestive of peptic injury. Gastric tissue, similarly, exhibited mild reactive changes. PCR analysis of the tissue from both location did not identify any organisms.

Conclusions

The diagnostic criteria for Whipple disease require identification of the organism in the biopsy of the intestine. Alternatively, the diagnosis can also be based on foamy macrophages containing rod-shaped bacilli in affected tissues, particularly in the lamina propria of the small intestine; or molecular methods of detection. Tropheryma whipplei can rapidly become undetectable in response to antibiotic therapy. Therefore, it’s important for healthcare professionals to be aware of the limitations of diagnostic tests and to interpret results in the context of the patient’s clinical history and presentation.

Posters: Neurodegenerative: Alzheimer
35
The Down Syndrome Biobank Consortium: A perspective

Background

Individuals with Down syndrome (DS) have a partial or complete trisomy of chromosome 21, resulting in an increased risk for early-onset Alzheimer’s disease (AD)-type dementia. Of the genetic mutations that cause AD, people with DS represent the largest cohort. Currently, there is a knowledge gap regarding the underlying neurobiological mechanisms of DS-related AD (DS-AD), partly due to limited access to well-characterized brain tissue and biomaterials for research.

Methods

Here, we present an international consortium of brain banks focused on collecting and disseminating brain tissue obtained from persons with DS throughout their lifespan, named the Down Syndrome Biobank Consortium (DSBC).

Results

The DSBC consists of 11 biobanking sites located in Europe, India, and the USA, which combined have more than 100 DS cases and 150 controls. The overall aim is to be an international repository for the collection and distribution of postmortem DS brain, serum, plasma, and CSF to investigate the neurobiological mechanisms and generate novel biomarkers for AD in DS. The DSBC has developed harmonized protocols including minimum recommended brain areas to sample for, neuropathologic diagnosis, staging criteria and storage protocols. A secondary main objective is to promote the use of DSBC samples for research in the DS space. DSBC members are active DS researchers and promote the use of DSBC samples by external researchers. DSBC members review all tissue requests, including site specific local legal requirements. The DSBC also performs educational activities such as clinicopathological conferences.

Conclusions

The DSBC provides a valuable framework to collect brain tissue and biofluids from clinically well-characterized DS and control cases needed to enhance sample availability for DS researchers. We hope to increase collaborative investigations and to raise awareness in the importance of examining postmortem tissue from people with DS and DS-AD as well as increase the quality and quantity of scientific publications in this space.

36
Illicit opioid abuse, traumatic brain injuries, and neocortical Alzheimer’s disease neuropathology

Background

Several research studies have demonstrated opioid abuse (ex: heroin) is associated with increased hyperphosphorylation and aggregation of the neurodegenerative protein tau. However, research on younger illicit opiate users is inconsistent with studies focused on older prescription opioid users, where heavy prescription opioid use does not associate with greater Alzheimer’s disease (AD) neuropathology. Few studies of opioid use and AD pathology assess traumatic brain injuries (TBI) as a potential confounder, yet it is estimated that in patients with TBI 10-44% have a premorbid history of illicit drug use. Recognizing the complex relationship between opioid use, TBI, and AD pathology, herein we analyzed tau and beta-amyloid in the brains of illicit drug users and matched-controls.

Methods

2,844 cases were co-enrolled in the Rochester Epidemiology Project and Mayo Clinic Tissue Registry with a date of death between 1/1/05 and 6/18/16. Drug abuse and TBI code set macros were run on diagnostic billing code and death certificate datasets for all cases. 59 cases with an illicit drug abuse diagnosis as well as 59 age- and sex-matched controls without a drug abuse history were selected. Tau and beta-amyloid immunohistochemistry was performed on tissue sections from the frontal neocortex and hippocampus.

Results

Drug abuse cases included 41 men and 18 women and had an average age at death of 47.3 years (range 18.3 – 88.1). In comparing tau pathology and beta-amyloid pathology in the frontal cortex, hippocampus, or both regions to the presence or absence of drug abuse, opioid abuse, TBI, and sex, no statistically significance differences were observed.

Conclusions

Taken together, these preliminary results suggest drug abuse, including specifically opioid abuse, does not appear to be related to AD pathology in two distinct regions of the brain. In this cohort, age at death played a much larger role in the pathogenesis of tau and beta-amyloid pathologies.

37
Resistance and Resilience to Neuropathology in Cognitive SuperAgers

Background

SuperAgers are persons over 80 with at least normal-for-age global cognition. The Northwestern University SuperAging program was initiated in 2001 and follows SuperAgers with serial neuropsychological testing and brain donation. The goal of this study was to examine neuropathologic features of SuperAgers with a focus on those who showed relative cognitive stability versus decline.

Methods

Thirty-eight SuperAgers underwent annual study visits as part of a longitudinal research program. Study visits included neurocognitive testing, questionnaires, and interviews with study partners; cognitive and functional status were determined in consensus meetings. Average age at death was 91.63 years (range 82-101 years). Average length of follow-up was 12.6 years (range, 3 to 25 years). Autopsy consisted of standard sampling throughout the neuraxis, routine histopathology, thioflavin S, and immunohistochemistry for 4G8, AT8, phospho-TDP 43 (Ser409-410), α-synuclein, and p62. Assessment was per 2012 NIA-AA recommendations.

Results

Of 38 SuperAging participants, 20 showed cognitive stability (52.6%), 18 showed cognitive decline (47.4%). Stable SuperAgers (26.3%) were more often rated ‘B1’ score compared to those who declined (0%; (OR = 0.737, p < 0.05). Of those who declined, two-thirds had intermediate or greater ADNC; 45% of stable SuperAgers met criteria for intermediate ADNC. Those who declined tended to be rated as “A3” (50%), CERAD “frequent” by Thioflavin S (38.9%), showed Tau positive neuritic plaques (23.5%), and ARTAG (66.7%); though higher than decliners, these features did not differ significantly from stable.

Conclusions

Initial clinicopathological analysis of SuperAgers indicates resistance to proteinopathy and cognitive resilience despite proteinopathy. “High” ADNC was entirely absent. Half of stable SuperAgers had “intermediate” ADNC. Although there was a trend toward stable SuperAgers demonstrating less proteinopathy, neuropathology was not predictive of antemortem cognitive trajectory. More studies are needed to explore the biological basis for resistance and resilience in this population, as well as the biological drivers of cognitive decline with age.

38
Neuropathology of Down syndrome: ADNC, ARTAG, and PART

Background

Down syndrome (DS) is frequently associated with Alzheimer disease neuropathologic change (ADNC). However, studies assessing the full spectrum of neurodegenerative pathologies according to modern consensus and staging criteria are limited.

Methods

We examined 35 brains from 2-70 year-old subjects with DS autopsied between 1986 and 2023 and performed comprehensive neurodegenerative evaluation using contemporary neuropathologic criteria. All cases were stained with thioflavin-S, p-tau (AT8) and amyloid-beta (6E10). Alpha-synuclein and TDP-43 pathologies were assessed only in subjects older than 30.

Results

All subjects over age 33 (n = 18) had ADNC, which included 12 (67%) with a high level, 2 (11%) with an intermediate level, and 3 (17%) with a low level, while one case could not be assigned an ADNC level due to incomplete Braak staging. As expected, there was a correlation between age and Braak stage, Thal phase, and CERAD score. The vast majority of cases in this subgroup (15/18, 83%) also had cerebral amyloid angiopathy and a small subset (3/18, 17%) had comorbid aging-related tau astrogliopathy (ARTAG). One case of a 70 year-old subject showed “amygdala predominant” Lewy-related pathology. None of the cases demonstrated limbic predominant age-related TDP-43 encephalopathy. Among the subjects under age 32 (n = 17), 6 had no evidence of proteinopathy. The remaining 11 cases did not meet 2012 NIA/AA criteria for ADNC: 4 (24%) harbored only diffuse plaques (DPs), 2 (12%) showed only rare hippocampal pretangles/neurites, 4 (24%) had both DPs and pretangles/neurites, and one case of a 29 year-old demonstrated primary age-related tauopathy (PART), Braak stage IV, with concomitant ARTAG. The youngest subject with DPs was an 11-year-old female, while the youngest subject harboring pretangles/neurites was a 14 -year-old female.

Conclusions

Individuals with DS can develop DP and pretangle/neurite pathology as early as their teenage years, and consistently demonstrate well-developed ADNC in their mid 30s, with comorbid pathologies being relatively uncommon.

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Alzheimer disease and cadaveric human growth hormone therapy: A comparative neuropathologic study

Background

The causal relationship of cadaveric human growth hormone (cHGH) in the development of Creutzfeldt-Jakob disease is known. Whether Alzheimer disease (AD) pathologic changes may occur related to cHGH treatment is unknown.

Methods

We compared the results of neuropathology analyses and RT-QuIC assays obtained studying the brain of two individuals who died at age 57 and 56 having been affected by early onset dementia.

Results

Patient 1 was a 55-year-old woman, whose past medical history included cHGH therapy during childhood for growth hormone deficiency. She had a two-year history of cognitive decline. She presented with bilateral apraxia of the upper and lower extremities and was evaluated for dementia and parkinsonism. Multiple cerebrospinal fluid (CSF) analyses revealed elevated total and phosphorylated tau, presence of 14-3-3 protein, negative RT-QuIC for prion protein (PrP) and decreased ß amyloid (Aß). The clinical diagnosis was corticobasal syndrome. Patient 2 was a woman, who had a history of head trauma with a laceration of the head around the age of nine and developed cognitive decline at age 54. The clinical diagnosis was AD. She died at age 56. The brain weights were similar being 1011 (Patient 1) and 1009 (Patient 2) grams. Neuropathologic studies revealed AD pathology (A3, B3, C3) and Aß angiopathy of leptomeningeal and intraparenchymal vessels. Immunohistochemistry for PrP was negative. The neuropathologic differences between these cases were that Aß angiopathy was more severe in Patient 1 and that tau inclusions in glial cells within the white matter were present only in Patient 2. Tests of multiple brain regions from the two brains by two different RT-QuIC assays that are broadly reactive with human PrP prion strains were negative. Tau RT-QuIC showed strong tau seeding activity in both brains.

Conclusions

A causal relationship between cHGH treatment and AD is not evident. Cryogenic electron microscopy studies are in progress.

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Building a Spatial Transcriptomic Atlas of Alzheimer’s Disease

Background

Investigation of Alzheimer’s disease (AD) biology is limited by its spatiotemporal heterogeneity. Numerous pathological features including neuritic plaques (NP), cerebral amyloid angiopathy (CAA), and neurofibrillary tangles (NFT) each demonstrate a spectrum of maturity across different brain regions, cortical layers, or individual cells within a single brain. Experiments performed on hom*ogenized tissue (bulk or single nuclei) cannot identify which molecular changes associate with each type and stage of pathology. Therefore, we aim to develop a spatial transcriptomic (ST) atlas of AD.

Methods

We evaluated prefrontal cortex samples from AD and control donors using 10x Genomics’ Xenium platform, which provides ST at single cell resolution with a customized panel of probes targeting 366 genes. pTau IHC (AT8) was performed on the same section used for ST, and A-beta IHC (6E10) was performed on a serial section and aligned. Computational annotation of IHC features facilitated comparison of genes differentially expressed in neurons and glia associated with each feature. Single nucleus multiomics (snRNA-seq + snATAC-seq) was performed on the same samples and integrated with published literature to support analysis of ST.

Results

ST identified about 100,000 cells per sample, including all cell types in expected proportions. Cells can be confidently classified and scored for behavioral programs, such as “Disease associated microglia”. Multiomic analysis generated data for over 2000 nuclei per sample with an average of 2,500 genes per nucleus, which cleanly aligned with published datasets. Scaling up the number of samples is ongoing, including multiple brain regions, Braak stages, and ApoE genotypes. Analyses performed on existing data include comparison of neurons with and without NFTs; glial cells associated with NFTs, CAA, and NPs; and spatial distribution of transcripts from specific genes.

Conclusions

We have developed an effective workflow for integrated ST and multiomics in AD, and are scaling up to develop a comprehensive spatial atlas.

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Amyloid Clearance, Alzheimer’s Disease Neuropathologic Change, and Comorbid Pathology in Four Patients Treated with Aducanumab

Background

Aducanumab is a monoclonal antibody targeting amyloid-β as disease-modifying strategy for Alzheimer’s disease (AD). Studies have shown high-dose aducanumab effectively removes amyloid-β as measured by amyloid-PET imaging; however, limited clinicopathologic correlations are available to characterize these novel therapies. We present autopsy findings of four treated patients, the challenges of interpreting the pathology with differing extent of treatments and confounding pathologies, and the clinical correlates.

Methods

Case 1: Male with a five-year history of AD. APOE genotype 4/4. Received aducanumab for 32 months and died 18 months after discontinuation. Case 2: Male with a seven-year history of AD. APOE genotype 3/4. Received placebo therapy before transitioning to aducanumab for 46 months and died three months after discontinuation. Case 3: Female with a nine-year history of AD. APOE genotype 3/4. Received aducanumab for 22 months and died 41 months after discontinuation. Case 4: Male with a seven-year history of AD. APOE genotype 3/4. Received aducanumab for 36 months and died 14 months after discontinuation. Patients 1 and 4 showed evidence of amyloid related imaging abnormalities (ARIA) on brain MRI during trial therapy.

Results

Neuropathologic examination of all four patients demonstrated advanced AD neuropathologic change (A3, B3, C3), moderate to severe cerebral amyloid angiopathy (CAA) without evidence of hemorrhage, and histopathologic features of amyloid clearance described in previous reports. Three of four patients showed clinically relevant comorbid pathology: Case 1, limbic (transitional) Lewy body disease; Case 2, diffuse neocortical Lewy body disease; and Case 3, limbic-predominant age-related TDP-43 encephalopathy (LATE), stage 2.

Conclusions

Four patients treated with aducanumab had histopathologic features of amyloid clearance but declined cognitively during and after treatment and showed advanced AD neuropathologic change. Time on therapy, interval to autopsy, and comorbid pathology confound the evaluation of therapeutic impact on the pathological and clinical diagnoses.

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CAPRIN1 expression and colocalization in human cortical neurons of Alzheimer’s disease

Background

The amyloid cascade of production of amyloid β (Aβ) peptides from amyloid-β precursor protein (APP) is the central event in the pathogenesis of Alzheimer’s disease (AD), although the mechanism remains unclear. Recently, cytoplasmic activation/proliferation-associate protein 1 (CAPRIN1) has been reported in neurodegeneration. Tracking CAPRIN1 through cellular organelles in human neurons from AD patient-derived induced pluripotent stem cells (iPSCs), we have demonstrated CAPRIN1 mediates APP degradation through the endosome-lysosome system and thereby regulates Aβ production in AD neurons. Here we report CAPRIN1 expression and its colocalization with APP in the neurons of AD brains.

Methods

AD autopsy brains and age-matched normal brains were obtained from Pathology Laboratory of Indiana University Health. Immunohistochemistry using CAPRIN1 and APP antibodies was performed on cerebral cortex, hippocampus, thalamus, basal ganglia, cerebellum, and brain stem. The expression levels of CAPRIN1 and APP in neurons were quantified. In addition, double immunofluorescence was performed and analyzed by confocal microscopes for colocalization of the proteins in neurons.

Results

Immunohistochemistry revealed that CAPRIN1 is predominantly expressed in the cytoplasm of neurons in normal and AD brains. CAPRIN1 is barely expressed in astrocytes, oligodendrocytes, and endothelial cells of blood vessels. Double immunofluorescence confocal microscopes revealed colocalization of CAPRIN1 and APP in the cytoplasm of the neurons of normal and AD brains. Further qualifications of CAPRIN1 expression will determine the difference of CAPRIN1 expression in neurons in different regions of normal and AD brains.

Conclusions

In this preliminary study, we demonstrated that CAPRIN1 is predominantly expressed and colocalized in the cytoplasm of neurons in AD brains, suggesting the role of CAPRIN1 in APP degradation in the neurons. Further large numbers of normal and AD brain will be analyzed to define the role of CAPRIN1 in AD pathogenesis.

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Investigating lysosomal autophagy pathway dysfunction in tau and TDP-43 synergy

Background

Although Alzheimer’s disease (AD) is pathologically defined by the presence of both amyloid plaques and neurofibrillary tangles, TDP-43 pathology often occurs co-morbidly with AD and correlates with more rapid cognitive decline and faster rates of hippocampal atrophy. We have previously shown that there is a synergistic relationship between tau and TDP-43 in a novel C. elegans model of combined tau and TDP-43 proteotoxicity, however the mechanisms that drive this interaction are unknown.

Methods

To further explore this relationship, we evaluated transcriptomic changes at time-points preceding frank neuronal loss in our C. elegans model of tau and TDP-43 co-expression (tau+TDP-43 Tg). We then followed up our findings with functional assessments in C. elegans as well multiplexed proteomics in human tissue using NanoString GeoMx Digital Spatial technology to relate findings in the model system back to the human disease.

Results

In the C. elegans transcriptomic analyses we found significant differential expression of genes enriched in the lysosomal-autophagy pathway. One of the most significantly differentially expressed genes with a human hom*ologue, cpr-8 (cathepsin-B), is a lysosomal enzyme involved in protein degradation. Subsequent function studies revealed that loss of cpr-8 function phenocopied the tau+TDP-43 phenotype in the tau strain. In our human NanoString experiment we compared neuron and astrocyte protein expression levels in the amygdala from donors with high AD pathology and co-morbid TDP-43 pathology to donors with high AD pathology only. Focusing on a panel of autophagy-related proteins we found significant differences in protein expression in AD+TDP-43 compared to AD alone.

Conclusions

Overall, these results highlight the relevance of lysosomal autophagy pathway dysfunction in the synergistic proteotoxicity of tau and TDP-43. Future work is necessary to further explore the specific mechanisms and timing of this dysfunction.

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Cerebrovascular Lesions in Patients with Neurodegenerative Disease: A Single-Center Autopsy Experience

Background

Cerebrovascular and neurodegenerative diseases are different in their primary mechanisms and manifestations, but their interplay can have repercussions on clinical diagnoses, prognosis, and therapy. There is growing interest in characterizing cerebrovascular lesions in neurodegenerative disease, in particular in patients with neurodegenerative diseases other than Alzheimer disease (AD).

Methods

We retrospectively reviewed all brain autopsies performed at UCLA between 2014 and 2022 that showed neurodegenerative features and characterized cerebrovascular lesions including atherosclerosis, arteriolosclerosis, cerebral amyloid angiopathy (CAA), infarcts, and hemorrhages. Cases with Braak stage V/VI were classified as pure AD (pure-AD), or mixed AD (mix-AD) based on the absence/presence of other neurodegenerative diagnoses. Cases with Braak stage IV or lower were classified as minimal AD (min-AD) or other neuropathological diagnosis (non-AD) based on the absence/presence of other neurodegenerative diagnoses.

Results

A total of 396 autopsy cases with neurodegenerative features were identified: 163 (41%) were pure-AD (age range: 29-114, mean: 78 years), 70 (18%) min-AD (age range: 59-111, mean: 70 years), 36 (9%) mix-AD (age range: 53-103, mean: 78 years), and 127 (32%) non-AD (age range: 58-98, mean: 71 years). CAA was observed more frequently in pure-AD and mix-AD (87% and 75%, respectively) than in min-AD and non-AD (40% and 25%, respectively). Severe arteriolosclerosis was observed more frequently in pure-AD (14%), min-AD (19%) and mix-AD cases (11%) compared to non-AD cases (3%). Macro-infarcts were observed more frequently among min-AD cases (26%) compared to other groups (pure-AD 11%, mix-AD 8%, non-AD 12%). Mild-to-moderate arteriolosclerosis, atherosclerosis, microinfarcts, vascular calcifications, aneurysms, and hemorrhages were observed in similar proportions between groups.

Conclusions

Cerebrovascular pathology is frequent in patients with neurodegenerative diseases, with CAA more common in those with advanced AD neuropathologic changes and macro-infarcts more common in those with minimal AD. Detailed characterization of cerebrovascular lesions is important in understanding the contribution of comorbid pathology in neurodegenerative diseases.

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Microglial MS4A gene SNPs are Associated with Cognitively Resilient Individuals in the Mayo Clinic Study of Aging

Background

The relationship between cognition and Alzheimer’s disease neuropathologic change (ADNC) is complex with a disconnect between cognition and ADNC in a subset of cognitively resilient individuals. Cognitive resilience is influenced by a multitude of mechanisms including genetics, sex, lifestyle, and education levels; however, the association between cognition, neuroinflammation , particularly microglia and microglial-specific genes, is understudied.

Methods

In Mayo Clinic Study of Aging (MCSA), we evaluated microglial-specific genes of interest, including MS4A gene cluster single-nucleotide polymorphisms (SNPs) in two analyses. 1) For those in the MCSA brain bank, we utilized antemortem global cognition z-scores, computed from memory, language, executive function, and visuospatial domain z-scores, to define cognitively intact (CI) participants as those performing in the upper quartile of their respective age groups. From the CI group, cognitively resilient (CR) individuals were identified as those with intermediate to high ADNC based upon neuropathologic evaluation. 2) In those with concomitant global cognition and amyloid PET data, we fit several parsimonious models to evaluate global cognition as a function of age, sex, amyloid PET, and SNPs of interest.

Results

CI individuals (n = 118) comprised 23% of the MCSA brain bank (total n = 505), with global z-scores mean -0.211. After neuropathologic evaluation, 35/118 (30%) showed no ADNC, 44/118 (37%) low ADNC, 30/118 (25%) intermediate ADNC, and 9/118 (8%) high ADNC, with CR individuals defined as intermediate to high ADNC (39/118, 33%). Of those CR individuals, 11 of 39 (28%) harbored an MS4A6A SNP and showed a protective effect on cognition over time in the amyloid-PET sample (p = 0.043).

Conclusions

In MCSA, CR individuals accounted for 7% of total cases with autopsy, which is in keeping with other published proportions. Moreover, microglial specific MS4A6A SNP, which plays a role in modulating microglial activation, was seen in approximately a third of resilient individuals, underscoring the complex interplay between microglia, genetics, and resilience.

46
Data-Driven Clinicopathologic Characterization of Parkinsonism and Nigral Neuron Loss in Alzheimer’s Disease