The diagnostic evaluation of progressive, multifocal sensory neuropathy can be challenging and prone to misdirection without a broad differential diagnosis and a careful clinical history. We present a 41-year-old healthy man with insidious onset of right foot paresthesia, which gradually extended to his left foot and right hand over several months. This case illustrates a systematic clinical reasoning approach to multifocal neuropathy, culminating in the unexpected diagnosis of a treatable cause for neuropathy. The case underscores the importance of maintaining diagnostic openness to rare but treatable causes in the evaluation of atypical neuropathy presentations.

RAS/RAF/MAPK signaling pathway is one of the best-defined cancer signaling pathways but its role in renal tumorigenesis is unknown outside of papillary renal neoplasm with reverse polarity (PRNRP), which harbors recurrent KRAS alteration. In 383 renal tumors with NGS performed at the University of Chicago and 406 tumors from the available TCGA PRCC/chromophobe RCC data sets, 6 and 9 renal tumors with RAS/RAF/MAPK pathway alteration were identified, respectively. KRAS was the most common gene to be altered (11/15) but alterations in BRAF (2/15), RAF1 (1/15), and NRAS (1/15) were also present. On the basis of morphology, the tumors were separated into 3 groups: classic PRNRPs (group 1), predominantly tubulocystic (group 2), and papillary with high-grade features (group 3). Although morphologically different, groups 1 and 2 shared many similarities in having (1) low-grade appearing eosinophilic tumor cells, (2) identical IHC profile (GATA3+/CK7+/CD117-/Vimentin-), (3) isolated KRAS alteration with no copy number variations, and (4) no proven metastatic potential. Group 3 showed predominantly papillary architecture composed of tumor cells with clear-to-eosinophilic cytoplasm and high-grade cytologic features. Unlike Group 1/2, 57% (4/7) of group 3 tumors showed additional gene alterations on top of RAS/RAF/MAPK pathway alteration and all group 3 tumors (7/7) showed significant copy number variations. On follow-up, 2 of the 7 (2/7) group 3 tumors have metastasized. One tumor with NRAS alteration showed unique morphology unlike any other tumors, composed of mixed tubulocystic and solid architecture with eosinophilic tumor cells. This tumor also showed significant copy number variations. The tumor was staged as pT4N1, displaying metastatic potential. This study shows that renal tumors with RAS/RAF/MAPK pathway alteration are heterogeneous morphologically, immunohistochemically, and molecularly. Although rare, recognition of this novel mechanism in renal tumorigenesis may be clinically important, as there are FDA-approved therapies that can target the RAS/RAF/MAPK pathway hyperactivation.

Background:Methylation class pleomorphic xanthoastrocytoma (mcPXA) comprises tumors with the DNA methylation signature of classical PXA but with a wider histologic spectrum, including overlap with glioblastoma (GBM). Methods:To clarify the histologic and molecular scope of mcPXA and characterize its clinical behavior, a cohort of 469 tumor samples from 458 patients matching to mcPXA by the DKFZ classifier (v12.6 score ≥0.85) was interrogated. Results:Patient median age was 23 years (range 1-73 years) with a female predominance (259 female/199 male). CDKN2A/B homozygous deletion was observed in 406 of 469 (87%) samples. In samples tested for BRAF p.V600E mutations (n = 279), 240 (86%) harbored the mutation. A chr7+/chr10- pattern was observed in 103 of 469 (22%) samples. Among samples tested for TERT promoter mutations (n = 143), 32 (22%) harbored the mutation. Progression-free and overall survival of patients with mcPXA were comparable to patients with methylation class IDH-mutant astrocytoma, low grade, but a GBM-like subset (ie, cases with a pre-methylation working diagnosis of GBM) showed shorter survival. Histologic features of high grade, including palisading necrosis and microvascular proliferation, were prognostic in mcPXA. Compared to patients with BRAF p.V600E-altered GBM, patients with mcPXA were younger and had a lower frequency of TERT promoter mutations. Conclusion:Tumors in mcPXA share molecular characteristics with histologically defined PXA, and high-grade histologic features can help predict their clinical behavior. The use of an epigenetic classification of PXA reveals that this group of tumors is more common than previously appreciated and warrants in-depth study to identify efficacious therapeutic options.

Introduction: Mouse models have been a valuable tool for studying the molecular mechanisms driving cerebral cavernous malformation (CCM) pathogenesis. However, genetically modified mouse models do not fully recapitulate human CCM disease, and endothelial dysfunction is implicated in multiple disease processes of the brain. Herein, we provide a radiation-induced platform of endothelial dysmorphism and physiologic dysfunction similar to that observed in CCM disease. By analyzing gene expression and pathway alterations in endothelial cells, this model enhances our understanding of the fundamental biological processes at play that are common and different between CCM and endothelial leak. Methods: Ten-week-old female C57BL/6 mice underwent irradiation with 40 Gy (@50% isodose) to a 5mm target within the left cerebral hemisphere. Brain parenchymal changes were assessed using contrast-enhanced CT imaging and histology. Endothelial cell RNA extraction and transcriptomic analysis were performed on formalin-fixed, paraffin-embedded coronal tissue sections from radiated and control hemispheres using the a digital spatial profiler system. Ingenuity Pathway Analysis (IPA) was employed to identify affected molecular pathways. Results: CT imaging revealed increased blood-brain barrier permeability within the irradiated area. Histologic analysis identified telangiectasias and microhemorrhages, consistent with dysmorphic endothelium and capillary malformations. Gene expression profiles between radiated vs. non-radiated endothelium demonstrated a clear distinction by principal component analysis. Differentially expressed genes were primarily associated with extracellular matrix organization, regulation of the actin cytoskeleton, and pro-inflammatory cytokine response pathways. Upstream analysis predicted TGFB1, TNF, lipopolysaccharide, dexamethasone, and angiotensinogen as potential regulators underlying the observed changes. Conclusion: This study elucidates a signature of dysmorphic endothelium in a non-genetically modified mouse model, revealing potential key molecular pathways and regulators that lead to endothelial leak. This model can guide mechanistic-based experimental studies and provide a platform for targeted interventions directed at critical components of CCM pathophysiology and the blood-brain barrier.

BACKGROUND:NTRK-rearranged spindle cell neoplasms constitute a novel, heterogeneous group of mesenchymal neoplasms originally described predominantly in soft tissue locations. They are commonly characterized by co-expression of S100 and CD34 immunostains and presence of NTRK fusions. While exceedingly rare, there are increasing reports of this lesion involving the gastrointestinal tract, presenting predominantly as large masses of the stomach, small bowel and colorectum. CASE PRESENTATION:We present a case of a 37-year-old male who on colonoscopy was found to have a one cm polyp of the sigmoid colon which was removed by hot snare polypectomy. Histologic examination revealed haphazardly arranged bland spindle cells with diffuse CD34 and S100 co-expression. A targeted Next-Generation RNA Fusion Assay identified a TPR::NTRK1 fusion, confirming the diagnosis of low-grade NTRK-rearranged spindle cell neoplasm. The mucosal and deep margins were free of tumor. In contrast to the previously reported cases, the patient was managed with polypectomy and active surveillance, and remained disease-free at 14 months follow up. CONCLUSION:This case contributes to the limited body of literature on gastrointestinal low-grade NTRK-rearranged spindle cell neoplasms and raises the possibility of endoscopic treatment consideration for carefully selected patients.

Introduction: Cerebral microbleeds (CMBs) are age-related manifestations of microangiopathies predisposing patients to hemorrhagic stroke (HS) and cognitive decline. Little work has currently been done to elucidate mechanisms in the pathophysiology of CMBs. Herein, we report transcriptomic signatures of CMBs, as well as important location/etiological and sex-specific differences, to glean mechanistic insights in human autopsy tissue and a murine E4FAD model. Methods: Fourteen human CMB lesions were identified at autopsy by a neuropathologist (n=9 lobar and n=5 deep brain lesions). In addition, 6 CMB lesions were identified from E4FAD mice (n=3 male and n=3 female) sacrificed at 8 months. For each lesion, contralateral control brain tissue was also obtained from the same brain. Serial sections were obtained for each lesional and control region. The first section was stained for non-heme iron to localize CMB and control regions. The second section was then used for paired GeoMx bulk spatial transcriptomics. Downstream differential expression analyses were completed using DeSeq2, with enriched pathway analyses using Ingenuity pathway analysis (IPA). Results: Spatial transcriptomics identified 781 differentially expressed genes (DEGs) in human CMB and 448 in E4FAD mouse lesions compared with controls (p<0.05, FDR corrected). Pathway enrichment identified 110 homologous pathways between human and E4FAD mouse CMBs, related to vascular permeability, senescence and neuroinflammation (p<0.05, FDR corrected). Furthermore, lobar compared to deep microbleeds showed 52 IPA pathways related to similar functions, particularly in relation to extracellular matrix deposition (p<0.05, FDR corrected). Notable differences identified etiologic-specific pathways, such as arteriolosclerosis and amyloid processing. Male compared with female mouse CMBs identified 12 common mechanisms involved in blood brain barrier dysfunction and neuroinflammation with differences related to sex-specific neurotransmitter and hormonal variances (p<0.05, FDR corrected). Conclusions: This is the first report to address transcriptomic changes within the CMB microenvironment, while addressing mechanistic differences by sex and lesional location. Further studies shall aim to delineate mechanisms that lead CMBs to develop into larger HS. The results also pave the way for identification of mechanistically linked circulating molecules in biomarker discovery as well as for potential therapeutic targets.

Stereotactic radiosurgery (SRS) is a procedure that delivers high-dose single fraction, targeted radiation to treat brain pathologies. Brain radiation necrosis is a significant side effect of SRS, resulting in severe clinical sequelae such as seizure, hemorrhage, stroke, and neurological deficit. While focused radiation causes DNA damage and cell death, radiation necrosis is mostly mediated by vascular injury. Yet the effects of SRS on the neurovascular unit (NVU) cells—microglia, astrocytes, and endothelial cells—remain poorly understood. This study establishes a mouse SRS model using 15 to 60 Gy to characterize NVU stress, providing histological and transcriptomic profiles of radiation-induced damage. Our findings demonstrate blood-brain-barrier (BBB) disruption, inflammatory cell infiltration, and microvascular pathology. Spatial transcriptomics identified differentially expressed genes and cell-cell communication across NVU components, revealing a coordinated stress response involving immune modulation, barrier integrity, and tissue remodeling pathways. This model provides a mechanistic framework for developing strategies to mitigate BBB and NVU stress.

HGAP was first described in 2018. However, prognostic factors of clinical outcome are not well-understood secondary to recent recognition and paucity of data for this diagnostic entity. Here, we utilize a large HGAP cohort (n=252) to survey the genomic landscape and explore prognostic correlates. NIH DNA methylation profiling was performed to identify HGAP cases and combined with publicly available datasets. Evaluable molecular markers, patient demographics, tumor location, imaging reports, treatment history (e.g. temozolomide, targeted therapy, surgery, and radiation) and survival data were assessed. Kaplan-Meier analysis was performed. The cohort included cases that matched to HGAP on the NIH/Bethesda methylation classifier at ≥0.9 confidence score. More males (60%) than females (40%) were in the cohort. Twenty-one percent of patients were known to have neurofibromatosis type 1. Posterior fossa location was predominant (57%, n=131). Common genomic findings included alterations in NF1 (58%), ATRX (50%), FGFR1 (15%), TP53 (9%) and PIK3CA (8%). CDKN2A/B homozygous deletion was identified in 80% of cases. MGMT promoter methylation was found in 55% of cases. Median progression free survival (mPFS) was 24 months (mo), and median overall survival (mOS) was 108 mo. Central histological review revealed 67% of cases were high-grade. High-grade histopathology (brisk mitotic activity) was not associated with survival. Immunohistochemical ATRX loss was associated with shorter mPFS (18.0 mo vs 35.5 mo, p=0.04) and mOS (93.7 mo vs not reached, p=0.04). The presence of ATRX genetic alterations was associated with shorter mOS (30 mo vs not reached, p=0.008). CDKN2A/B homozygous deletion and MGMT status were not correlative with patient outcome. HGAP is a glial neoplasm that shows frequent tumor recurrence. The majority of HGAP cases are high-grade. Our analysis of correlates with patient outcome suggests immunohistochemical ATRX loss and molecular ATRX alteration may be important poor prognostic markers.

Modern pathology requires precise data interpretation to enhance patient care and operational efficiency. We applied a Python-based informatics approach to analyze turnaround times, second opinion consultations, and diagnostic concordance. This investigation aimed to determine actionable insights and assess the feasibility of a subspecialty frozen section service. Monthly frozen section data from 2023 was gathered. A Python script was developed to transform the data into over 150 dynamic graphs. These visualizations detail turnaround times, second opinions, concordance rates, and overall performance metrics. Breakdowns by case complexity and specialty pairing (surgeon and pathologist subspeciality) further informed our analysis. The average turnaround time of 43.4 minutes for same-specialty pairs versus 46.9 minutes for different specialties. Cases requiring a second opinion extended turnaround time notably, and delays were most pronounced in breast, gastrointestinal, and renal cases. Diagnostic concordance remained high at 97.5% regardless of surgeon-pathologist speciality pairing. Our findings demonstrate that aligning surgeon and pathologist specialties modestly reduces turnaround times, yet complexity and second opinion requirements significantly extend them. Tailored second opinion protocols and subspecialty-specific strategies can optimize efficiency and maintain high diagnostic accuracy. Also, targeted interventions, such as refining second opinion protocols, can enhance both operational efficiency and diagnostic accuracy in modern pathology.

A putative molecular subtype of IDH-wildtype diffuse glioma with recurrent MAPK pathway alterations has recently been reported. By dimensionality reduction analysis of genome-wide methylation profiling, these tumors form a distinct methylation cluster of gliomas. Characterization of 47 tumors from 45 patients reveals that these gliomas are predominantly supratentorial in young adults, are highly infiltrative, and harbor mitogen-activated protein kinase (MAPK) pathway alterations with high rates of CDKN2A/2B deletion, PDGFRA amplification, MYCN amplification, NF1 variants, and BRAF alterations. The tumors' epigenetics are distinct from other adult and pediatric gliomas in the 2021 World Health Organization (WHO) classification. The histology of the gliomas most often demonstrates high-grade astrocytic features, but can be variable from tumor to tumor, as well as fall into a spectrum of histologic grades. Outcomes show considerable variability based on histologic grade and molecular features, supporting grading within this group of tumors to ensure optimal care choices on an individual patient basis. These unifying epigenetic, sequencing, and infiltrative astrocytic features allow the tumors to be considered diffuse astrocytoma, adolescent, and young adult-type, with MAPK alterations (DAYA).

Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we test a prevailing hypothesis that NE ruptures trigger the pathological cGAS-STING cytosolic DNA-sensing pathway using a mouse model of Lamin cardiomyopathy. The reduction of Lamin A/C in cardio-myocyte of adult mice causes pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures are followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remains inactive. Deleting cGas or Sting does not rescue cardiomyopathy in the mouse model. The lack of cGAS-STING activation is likely due to the near absence of cGAS expression in adult cardiomyocytes at baseline. Instead, extracellular matrix (ECM) signaling is activated and predicted to initiate pro-inflammatory communication from Lamin-reduced cardiomyocytes to fibroblasts. Our work nominates ECM signaling, not cGAS-STING, as a potential inflammatory contributor in Lamin cardiomyopathy.

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

10023 Background: Rapid and accurate identification of morphologic features of neuroblastic tumors (NTs) is critical for risk stratification and therapeutic decision making. The prognostic value of features like neuroblast differentiation, mitosis-karyorrhexis index (MKI), and Schwannian stromal presence is well established. Deep learning permits objective histopathological analysis, streamlining workflows for pathologists, notably in rare cancers. In rare cancers, our method minimizes bias and optimizes limited data using transfer and self-supervised learning (SSL) for feature extraction, with improved explainability. Here, we used an artificial intelligence-based model to morphologically classify NT tumors and MYCN-amplification. Methods: Annotated H&E-stained slides of diagnostic NT tumor biopsies from the University of Chicago and the Children’s Oncology Group were digitalized. Pathologists defined three binarized measures including diagnostic category (ganglioneuroblastoma/neuroblastoma), grade (differentiating/poorly differentiating), and MKI (low and intermediate/high). MYCN status was abstracted from patient records (amplified/non-amplified). Using Slideflow, our open-source pipeline, we developed an attention-based multiple instance learning model with features extracted by CTransPath, a SSL model pretrained on pan-cancer images from The Cancer Genome Atlas. For each measure, model performance was evaluated using 5-fold cross validation by aggregating k-fold model predictions across multiple metrics. Patients were excluded from a model if the measure of interest was unknown. Feature significance was assessed visually using Class Activation Mapping (Grad-CAM). Results: The mean age of the study cohort (n = 172) was 3.66 years. Of patients with clinical information, 84 of 138 (60.2%) had metastatic disease and 94 of 133 (70.7%) were high-risk. Of the 148 tumors with a diagnostic category of neuroblastoma, 93.2% were poorly differentiated and 25% had high MKI. Of the 135 tumors with known MYCN status, 40 were amplified (29.6%). The final models excelled across all outcomes, performing best for diagnostic category, grade, and MYCN status (Table 1). Physician review of the attention-based heatmaps for all measures highlighted biologically relevant regions such as neuropil. Conclusions: We created a deep learning pipeline for auto-characterization of digitized H&E-stained NT pathology slides. Our approach may also aid in identifying molecular features including MYCN-amplification. Review of heatmaps showed pertinent biological tissue, boosting model reliability.[Table: see text]

BackgroundRadiation treatment for diseases of the brain can result in hemorrhagic adverse radiation effects. The underlying pathologic substrate of brain bleeding after irradiation has not been elucidated, nor potential associations with induced somatic mutations.MethodsWe retrospectively reviewed our department's pathology database over 5 years and identified 5 biopsy specimens (4 patients) for hemorrhagic lesions after brain irradiation. Tissues with active malignancy were excluded. Samples were characterized using H&E, Perl's Prussian Blue, and Masson's Trichrome; immunostaining for B-cells (anti-CD20), T-cells (anti-CD3), endothelium (anti-CD31), macrophages (anti-CD163), α-smooth muscle actin, and TUNEL. DNA analysis was done by two panels of next-generation sequencing for somatic mutations associated with known cerebrovascular anomalies.ResultsOne lesion involved hemorrhagic expansion among multifocal microbleeds that had developed after craniospinal irradiation for distant medulloblastoma treatment. Three bleeds arose in the bed of focally irradiated arteriovenous malformations (AVM) after confirmed obliteration. A fifth specimen involved the radiation field distinct from an irradiated AVM bed. From these, 2 patterns of hemorrhagic vascular pathology were identified: encapsulated hematomas and cavernous-like malformations. All lesions included telangiectasias with dysmorphic endothelium, consistent with primordial cavernous malformations with an associated inflammatory response. DNA analysis demonstrated genetic variants in PIK3CA and/or PTEN genes but excluded mutations in CCM genes.ConclusionsDespite pathologic heterogeneity, brain bleeding after irradiation is uniformly associated with primordial cavernous-like telangiectasias and disruption of genes implicated in dysangiogenesis but not genes implicated as causative of cerebral cavernous malformations. This may implicate a novel signaling axis as an area for future study.

Background A significant unmet need exists for the treatment of glioblastoma, IDH-wildtype (GBM). Preclinical work shows that acetazolamide sensitizes GBM to temozolomide (TMZ) by overcoming TMZ resistance due to BCL-3-dependent upregulation of carbonic anhydrase. Acetazolamide is Food and Drug Administration-approved for the treatment of altitude sickness. Drug repurposing enables the application of drugs to diseases beyond initial indications. This multi-institutional, open-label, phase I trial examined a combination of acetazolamide and TMZ in patients with MGMT promoter-methylated high-grade glioma.Methods A total of 24 patients (GBM, IDH-wildtype = 22; Grade 4 astrocytoma, IDH-mutant = 1; Grade 3 astrocytoma, IDH-mutant = 1) were accrued over 17 months. All patients received oral acetazolamide (250 mg BID for 7 days increased to 500 mg BID for Days 8-21 of each 28-day cycle) during the adjuvant phase of TMZ for up to 6 cycles.Results No patient had a dose-limiting toxicity. Adverse events were consistent with known sequelae of acetazolamide and TMZ. In the 23 WHO Grade 4 patients, the median overall survival (OS) was 30.1 months and the median progression-free survival was 16.0 months. The 2-year OS was 60.9%. In total 37% of the study population had high BCL-3 staining and trended toward shorter OS (17.2 months vs N.R., P = .06).Conclusions The addition of acetazolamide is safe and tolerable in GBM patients receiving standard TMZ. Survival results compare favorably to historical data from randomized trials in patients with MGMT promoter-methylated GBM and support examination of acetazolamide in a randomized trial. BCL-3 expression is a potential biomarker for prognosis in GBM or for patients more likely to benefit from TMZ.

Background: Cerebral microbleeds (CMBs) are age-related vascular lesions, predisposing patients to a lifetime risk of hemorrhagic stroke and cognitive decline. The cellular architecture in the lesions and their genetic alterations have not been elucidated, limiting biomarker development and therapeutic targeting. Herein, we report histopathological and transcriptomic signatures of CMBs in a murine model. Methods: Seven APOE4 + / + ; 5xFAD mice were sacrificed at 8 months and had their brains harvested. Brain tissue was stained with H&E and Prussian Blue for non-heme iron to identify CMBs. Astrocytes (GFAP + ), microglia (CD163 - /Iba1 + ), macrophages (CD163 + /Iba1 + ), B cells (CD20 + ), T cells (CD3 + ), and endothelium (CD31 + ) were stained using multiplexed immunofluorescence. Regions of interest (ROIs) around CMBs and directly adjacent tissue, as well as contralateral control brain tissue, were selected and quantified using QuPath. GeoMx spatial transcriptomics was performed on lesions and contralateral control brain areas in the same mice. The DEG analysis was completed using DESeq2. Results: CMBs had higher astrocyte and microglia counts (normalized to ROI area) than control tissue (p<0.05). Microglia showed reactive, amoeboid morphological changes: hypertrophic cell bodies and shorter processes. T cells had a trend toward higher cell counts in CMBs (p=0.12). Macrophages and B cell counts were similar between CMBs and controls. The spatial transcriptomics identified 19 DEGs between CMBs and control tissue (p<0.1, FDR corrected) related to permeability, cell metabolism, apoptosis, and transcriptional regulation. Conclusions: This is the first report relating the CMB lesional microenvironment to spatial transcriptomic analyses. Further studies shall aim at confirming these findings in human CMB lesions, and their association with circulating molecules. The results pave the way for enhanced mechanistic insight, biomarker discovery and potential therapeutic targeting.