Autopsy Tissue Research Articles

NIMG-58. MACHINE LEARNING-GENERATED WHOLE-BRAIN RADIO-PATHOMIC TUMOR PROBABILITY MAPS WITHIN CONTRAST-ENHANCEMENT STRATIFY SURVIVAL IN GLIOBLASTOMA PATIENTS UNDERGOING GROSS-TOTAL RESECTION

Abstract BACKGROUND Even for those undergoing gross total resection (GTR), patients with WHO grade 4 glioblastoma (GBM) have poor overall survival (OS). Stratifying those who will have better OS vs. those that will not remains challenging, with conventional imaging and radiomic models falling short. This study utilizes machine learning (ML)-generated radio-pathomic models based on autopsy tissue as ground-truth to enhance prognostication through tumor probability maps (TPM) within preoperative contrast-enhancing (CE) regions of GBM. METHODS Data from the UCSF-PDGM-v2 and UPenn-GBM databases were analyzed, focusing on patients who underwent GTR with WHO 4 IDH-wildtype glioblastoma (n = 528). Preoperative tumor cellularity within CE was quantified using ML-generated TPMs, with a median threshold of 0.7242. Kaplan-Meier survival curves were employed to compare OS between patients with low (≤ 0.7242) and high (> 0.7242) TPM values in CE regions. Statistical significance was evaluated using log-rank tests. RESULTS Kaplan-Meier curves demonstrated significant stratification in survival outcomes based on TPM values. Patients with lower TPM values in CE regions had a median OS of 567 days, compared to 457 days for those with higher TPM values (log-rank p-value: < 0.001). These findings suggest that lower tumor cellularity in CE regions as assessed by TPMs correlates with improved survival, offering a more nuanced stratification than current MRI-based methods. CONCLUSION This study underscores the potential of TPM metrics as a powerful prognostic tool in GBM. The significant correlation between lower TPM values in CE regions and better median OS indicates that TPM can effectively stratify patients, aiding in clinical decision-making and personalized treatment planning. Future research is necessary to validate these findings in larger, diverse patient populations and to explore the biological mechanisms underlying these survival differences.

NIMG-59. COMPARATIVE ANALYSIS OF LONG-TERM GLIOBLASTOMA SURVIVORS USING MACHINE LEARNING GENERATED RADIO-PATHOMIC METRICS

Abstract BACKGROUND Glioblastoma (GBM) remains the most devastating central nervous system malignancy with poor overall survival (OS), even with gross total resection (GTR) and chemoradiation. Identifying factors associated with long-term survival (LTS) in GBM patients is challenging but crucial for improving prognostic models. This study compares an internal dataset of the top 10% long-term survivors of GBM (n=14) against a combined external cohort of 528 GBM patients who underwent GTR. METHODS We used a previously published machine learning-generated radio-pathomic model based on autopsy tissue as ground-truth to generate whole-brain maps of cell density and tumor probability maps (TPM) using conventional preoperative MRI sequences in both our internal dataset and a combined external cohort of GBM patients who underwent GTR from the UCSF-PDGM-v2 and UPenn-GBM databases. We then analyzed clinical and radio-pathomic metrics, including cell density and TPM values within contrast-enhancing (CE) and FLAIR hyperintense regions. Statistical comparisons were performed using t-tests. RESULTS Cell density metrics showed significant differences, with lower cell density in contrast-enhancing regions (t-value: 4.709, p<0.001; median: 1559 vs. 1959) and in FLAIR hyperintense regions (t-value: 3.585, p<0.001; median: 1356 vs. 1568) within the LTS group. TPM metrics also showed significant differences, with lower TPM values in contrast-enhancing regions (t-value: 3.312, p<0.001; median: 0.71 vs. 0.72) and in non-enhancing regions (t-value: 2.606, p<0.01; median: 0.68 vs. 0.69) in the LTS group. CONCLUSION Our comparative analysis reveals distinct radio-pathomic characteristics associated with long-term survival in GBM patients. Reduced cell density and lower TPM values in both contrast-enhancing and FLAIR hyperintense regions are significantly correlated with long-term survival. These findings suggest that integrating radio-pathomic data may enhance prognostic models and warrants further studies evaluating the relationship of these markers and OS.

Identification of a 5-gene signature panel for the prediction of prostate cancer progression.

Despite nearly 100% 5-year survival for localised prostate cancer, the survival rate for metastatic prostate cancer significantly declines to 32%. Thus, it is crucial to identify molecular indicators that reflect the progression from localised disease to metastatic prostate cancer. To search for molecular indicators associated with prostate cancer metastasis, we performed proteomic analysis of rapid autopsy tissue samples from metastatic prostate cancer (N = 8) and localised prostate cancer (N = 2). Then, we utilised multiple independent, publicly available prostate cancer patient datasets to select candidates that also correlate with worse prostate cancer clinical prognosis. We identified 154 proteins with increased expressions in metastases relative to localised prostate cancer through proteomic analysis. From the subset of these candidates that correlate with prostate cancer recurrence (N = 28) and shorter disease-free survival (N = 37), we identified a 5-gene signature panel with improved performance in predicting worse clinical prognosis relative to individual candidates. Our study presents a new 5-gene signature panel that is associated with worse clinical prognosis and is elevated in prostate cancer metastasis on both protein and mRNA levels. Our 5-gene signature panel represents a potential modality for the prediction of prostate cancer progression towards the onset of metastasis.

BCAS1-positive oligodendrocytes enable efficient cortical remyelination in multiple sclerosis.

Remyelination is a crucial regenerative process in demyelinating diseases, limiting persisting damage to the central nervous system (CNS). It restores saltatory nerve conduction and ensures trophic support of axons. In multiple sclerosis (MS) patients, remyelination has been observed in both white and grey matter and found to be more efficient in the cortex. Brain-enriched myelin-associated protein 1 (BCAS1) identifies oligodendrocyte lineage cells in the stage of active myelin formation in development and regeneration. Other than in the white matter, BCAS1+ oligodendrocytes are maintained at high densities in the cortex throughout life. Here, we investigated cortical lesions in human biopsy and autopsy tissue from patients with MS in direct comparison to demyelinating mouse models and demonstrate that following a demyelinating insult BCAS1+ oligodendrocytes in remyelinating cortical lesions shift from a quiescent to an activated, internode-forming morphology co-expressing myelin-associated glycoprotein (MAG), necessary for axonal contact formation. Noteworthy, activated BCAS1+ oligodendrocytes are found at early time points of experimental demyelination amidst ongoing inflammation. In human tissue, activated BCAS 1+ oligodendrocytes correlate with the density of myeloid cells, further supporting their involvement in an immediate regenerative response. Furthermore, studying the microscopically normal appearing non demyelinated cortex in patients with chronic MS, we find a shift from quiescent BCAS1+ oligodendrocytes to mature, myelin-maintaining oligodendrocytes, suggesting oligodendrocyte differentiation and limited replenishment of BCAS1+ oligodendrocytes in long-standing disease. We also demonstrate that part of perineuronal satellite oligodendrocytes are BCAS1+ and contribute to remyelination in human and experimental cortical demyelination. In summary, our results provide evidence from human tissue and experimental models that BCAS1+ cells in the adult cortex represent a population of pre-differentiated oligodendrocytes that rapidly react after a demyelinating insult thus enabling immediate myelin regeneration. In addition, our data suggest that limited replenishment of BCAS1+ oligodendrocytes may contribute to the remyelination failure observed in the cortex in chronic MS.

Abstract C122: Androgen receptor-mediated regulation of innate immunity in prostate cancer in Black men

Abstract Black men are two times more likely to die from prostate cancer (PCa) than White men. We reported that innate immune cell infiltration varies between races, and others have reported differences in androgen receptor (AR) expression by race or ancestry. Herein, we evaluated AR status and immune cell infiltration in primary and metastatic PCa tissues obtained from Black and White men. RNA in situ hybridization (RISH) AR analysis was done on a tissue microarray (TMA) constructed from radical prostatectomy tissues from 120 Black men matched to 60 White men on age, grade and stage. RISH or immunohistochemistry (IHC) analysis of AR, NKX3.1, prostate specific antigen (PSA), mast cells (tryptase), neutrophils (CD66), and macrophages (CD206, CD68, CD80) was done on a TMA containing rapid autopsy tissues from metastatic sites (n=11) collected from Black (n=5) and White (n=16) men. Multiplexing IHC (mIHC) was used to assess AR, neutrophils (CD66ce), mast cells (tryptase), T-cells (CD4, CD8), and M2 macrophages (CD163) on radical prostatectomy whole-tissue sections obtained at Johns Hopkins Medicine, Baltimore, MD and biopsy tissues obtained from the University of Nairobi, Nairobi, Kenya. Tumor regions on TMAs were annotated by a pathologist and signal intensity per unit area measured using TMAJ and FrIDA software. HALO Image Analysis Platform (version 3.6.4134.137; Indica Labs) was used to analyze mIHC stained tissues. Statistical analysis and graphing were done using GraphPad Prism. There was increased AR mRNA expression in primary cancer tissues from Black men compared to White men (p<0.0001) after negative binomial regression with the adjustment of TMA set, age, grade and stage. There was a negative correlation between AR expression and CD66+ neutrophils in tumor and benign tissues from Black men (R = -0.318;p = 0.002) but this correlation was not significant among White men (R= -0.045; p = 0.765). Interestingly, we measured a positive correlation between AR mRNA expression and tryptase+ mast cells among White men (R= 0.320; p=0.016), but a trend toward a negative relationship among Black men (R= -0.252;p = 0.075) We also measured a positive correlation between AR and CD163+(R= 0.324; p<0.001), CD68+(R= 0.514;p<0.001), and CD80+(R= 0.386;p<0.001) macrophages in primary tumor tissues from all men. NKX3.1 and PSA protein were significantly increased in metastatic tissues from White compared to Black men. Conversely, CD68+ and CD80+ macrophages were increased in Black compared to White men. CD66+ neutrophils and CD206+ macrophages were increased in AR- compared to AR intact metastatic tissues. Immune cells appeared to be primarily void of AR expression in primary cancer tissues by mIHC, but there was a subset CD4+ and CD8+ T-cell clusters that express AR. Immune cell infiltration varies by the presence of AR in both primary and metastatic PCa tissues. We observed differences in AR mRNA and AR-related proteins between Black and White patients which may contribute to the differences observed in immune cell infiltration between the two races Citation Format: Kennedy Rains, Katie M. Farkouh, Taylor N. Godwin, Eric Erak, Aakash Parikh, Michael Considine, Elana Fertig, Jiayun Lu, Charles Waihenya, Valerie Odero-Marah, Karen S. Sfanos, Angelo M. De Marzo, Corinne E. Joshu, Janielle P. Maynard. Androgen receptor-mediated regulation of innate immunity in prostate cancer in Black men [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C122.

Mouse Models for the Study of Borrelia burgdorferi Infection.

Lyme disease, a tickborne illness caused by Borrelia burgdorferi, is an emerging, significant public health concern. B. burgdorferi infections are challenging to study because of their complex life cycle that requires adaptation to both ticks and mammalian hosts for long-term survival and transmission. Bacterial adaptation is accomplished through extensive gene expression alterations in response to environmental cues that remain to be more fully explored. Mouse models of infection serve as valuable tools for studying B. burgdorferi adaptation to the mammalian host and the spirochete's ability to cause persistent infections and thus to interact with and evade the immune system. This article details three mouse models that differ in their primary methods of infection: infestation with B. burgdorferi infected ticks, intradermal inoculation of culture-grown spirochetes, and infection via subcutaneous transplantation of infected tissue. Each method offers unique advantages and limitations. Tick infestation is the route of natural transmission but presents logistical challenges. Syringe inoculation is easy and provides precise control over the infectious dose, but infection is with culture-adapted bacteria. Transplantation of infected tissue introduces mammalian-host-adapted B. burgdorferi in precise anatomical locations, but misses the transfer of tick factors affecting immunity. Detailed protocols are provided for each of the three infection routes, and pros and cons of each method are outlined to help researchers identify the best approach for a research question to be addressed. A protocol is also provided for the treatment of mice with antibiotics that reliably eliminates detectable spirochetes from the animals. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Syringe inoculation of mice with cultured B. burgdorferi and collection of necropsy tissues Basic Protocol 2: Infection of mice with B. burgdorferi via tick infestation Basic Protocol 3: Infection of mice with host-adapted B. burgdorferi via tissue transplant Support Protocol: Clearance of B. burgdorferi by antibiotic treatment.

Amanitin determination in bile samples by UHPLC-MS: LR-MS and HR-MS analytical performance

Consumption of misidentified foraged mushrooms containing bicyclic amanitin octapeptides is a worldwide public health and veterinary problem, being considered one of the deadliest accidental human and canine food ingestion due to acute liver failure (ALF). Reversal of advanced ALF and complete clinical recovery can be achieved following definitive removal of accumulated amatoxin laden bile from the gallbladder. An accurate means of quantifying amanitin content in aspirated bile is, therefore, urgently needed. Building on our prior work validating a method to detect and quantify amanitin in hepatic autopsy tissue, the development of an accurate method of measuring α- and β-amanitin in aspirated gallbladder bile was performed to evaluate the efficiency of this emergency procedure applied as a clinical treatment for intoxicated patients. A solid-phase extraction (SPE) procedure was optimized followed by detection based on ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). Low resolution mass spectrometry (LRMS) was compared with high resolution (HRMS) by the validation of UHPLC-MS/MS (triple quadrupole MS) and UHPLC-ToF-MS (time-of-flight MS). Both methods were able to detect amatoxins in bile with limits of detection and quantification ranging from 2.71 to 3.46 µg.kg-1, and 8.36–9.03 µg.kg-1 for α-amanitin and, 0.32–1.69 µg.kg-1 and 0.55–5.62 µg.kg-1 for β-amanitin, respectively. Validation was completed with the evaluation of linearity, specificity, robustness, recovery, and precision following the ICH guidelines and CIR 808/2021. The validated methods were finally applied to bile samples obtained 48–96 hours + post-ingestion from 4 amatoxin poisoning patients who underwent gallbladder drainage procedures in Vietnam, Canada, and California. Gallbladder bile from patients with amatoxin mushroom poisoning contained significant amanitin content, even when aspirated several days post-ingestion, thus confirming the important role of enterohepatic circulation in amatoxin hepatotoxicity. This work represents a high and unique analytical throughput in amanitin poisoning allowing to efficiently respond to this fatal health problem.

Molecular characteristics of fowl adenovirus strains detected in broiler chickens on diets without immunostimulant supplements.

Outbreaks of fowl adenovirus (FAdV) infection in chicken flocks in Poland threaten birds' health and lives and are rising in frequency. The risk of these infections in immunocompromised poultry flocks with developed clinical symptoms was analysed through virus detection in broiler chicks and correlation of cases with the birds' immune strength. Samples were analysed from four broiler farms with chicks from the same hatchery in Silesia, Poland where feeding regimes were different. A normal diet was provided to birds on the control farm; a normal diet and probiotic, prebiotic, vitamin and microelement supplementation was supplied on another farm; a normal diet and antibiotics on the third; and a normal diet and both forms of supplementation were given on the fourth farm. Amplification of the virus DNA in a PCR with hexon gene L1 loop hypervariable region 1-4 primers determined the molecular characteristics of isolates of adenovirus strains obtained from necropsy tissue samples. The amplicon sequences were analysed, the pair-wise distances were determined, the maximum likelihood estimate for the gamma parameter for site rates was produced, Tajima's D neutrality test was run and the relative synonymous codon usage and transition/transversion bias were calculated. Two species and two serotypes of fowl adenovirus - MW353018-FAdV-1/A-L-liver and MW353019-FAdV-5/B-I-intestine - were isolated in three-week-old broiler chicks on the control farm. Supplementation of broiler chicken flocks with probiotics, prebiotics, vitamins and microelements may have a significant beneficial effect on immunity and can prevent virus infection. The studies provided new information on the molecular characteristics of adenovirus strains isolated from chicks with a low level of immunity.

PITFALLS IN AUTOPSY TISSUE SAMPLING

Objective: To determine the pre-analytic factor which lead to autolysis in tissue sample thus hindering the establishment of histo-pathological cause of death in some autopsy tissue samples. Methods: Data collected from April, 2017 to September, 2018 (18 mo) at Department of Pathology, Dr. S. N. Medical College, Jodhpur. Results: The study, conducted over 18 mo from April 2017 to September 2018 at the Department of Pathology, Dr. S. N. Medical College, Jodhpur, analyzed 637 autopsy cases. These cases were predominantly from urban areas (70.17%), with a lesser number from rural regions (29.83%). A total of 698 tissue samples were received in various containers, with the majority (95.71%) preserved in formalin. The examination revealed that 38.4% of the samples were not autolysed, 36.1% were partially autolysed, and 25.5% were completely autolysed. The findings highlight a significant impact of the pre-analytic phase on tissue sample quality, notably the interval between death and autopsy, which was less than 24 h in 86.19% of cases, and the time from autopsy to histopathological examination, which ranged up to 1044 d. These factors were pivotal in the preservation status of the tissues, with formalin proving to be the most effective fixative in preventing autolysis, thus facilitating the accurate establishment of histopathological causes of death. Conclusion: If autopsy specimens are submitted for histopathological examination with proper protocol of transfer to fixative solution and transportation of tissue, this will help in determining the cause of death in majority of the autopsy specimens, therefore avoiding false negative results.

Characterization and modulation of the pro-inflammatory effects of immune cells in the canine intervertebral disk.

Intervertebral disk (IVD) degeneration affects both humans and canines and is a major cause of low back pain (LBP). Mast cell (MC) and macrophage (MØ) infiltration has been identified in the pathogenesis of IVD degeneration (IVDD) in the human and rodent model but remains understudied in the canine. MC degranulation in the IVD leads to a pro-inflammatory cascade and activates protease activated receptor 2 (PAR2) on IVD cells. The objectives of the present study are to: (1) highlight the pathophysiological changes observed in the degenerate canine IVD, (2) further characterize the inflammatory effect of MCs co-cultured with canine nucleus pulposus (NP) cells, (3) evaluate the effect of construct stiffness on NP and MCs, and (4) identify potential therapeutics to mitigate pathologic changes in the IVD microenvironment. Canine IVD tissue was isolated from healthy autopsy research dogs (beagle) and pet dogs undergoing laminectomy for IVD herniation. Morphology, protein content, and inflammatory markers were assessed. NP cells isolated from healthy autopsy (Mongrel hounds) tissue were co-cultured with canine MCs within agarose constructs and treated with cromolyn sodium (CS) and PAR2 antagonist (PAR2A). Gene expression, sulfated glycosaminoglycan content, and stiffness of constructs were assessed. CD 31+ blood vessels, mast cell tryptase, and macrophage CD 163+ were increased in the degenerate surgical canine tissue compared to healthy autopsy. Pro-inflammatory genes were upregulated when canine NP cells were co-cultured with MCs and the stiffer microenvironment enhanced these effects. Treatment with CS and PAR2 inhibitors mediated key pro-inflammatory markers in canine NP cells. There is increased MC, MØs, and vascular ingrowth in the degenerate canine IVD tissue, similar to observations in the clinical population with IVDD and LBP. MCs co-cultured with canine NP cells drive inflammation, and CS and PAR2A are potential therapeutics that may mitigate the pathophysiology of IVDD in vitro.

Infection with SARS-CoV-2 can cause pancreatic impairment

Evidence suggests associations between COVID-19 patients or vaccines and glycometabolic dysfunction and an even higher risk of the occurrence of diabetes. Herein, we retrospectively analyzed pancreatic lesions in autopsy tissues from 67 SARS-CoV-2 infected non-human primates (NHPs) models and 121 vaccinated and infected NHPs from 2020 to 2023 and COVID-19 patients. Multi-label immunofluorescence revealed direct infection of both exocrine and endocrine pancreatic cells by the virus in NHPs and humans. Minor and limited phenotypic and histopathological changes were observed in adult models. Systemic proteomics and metabolomics results indicated metabolic disorders, mainly enriched in insulin resistance pathways, in infected adult NHPs, along with elevated fasting C-peptide and C-peptide/glucose ratio levels. Furthermore, in elder COVID-19 NHPs, SARS-CoV-2 infection causes loss of beta (β) cells and lower expressed-insulin in situ characterized by islet amyloidosis and necrosis, activation of α-SMA and aggravated fibrosis consisting of lower collagen in serum, an increase of pancreatic inflammation and stress markers, ICAM-1 and G3BP1, along with more severe glycometabolic dysfunction. In contrast, vaccination maintained glucose homeostasis by activating insulin receptor α and insulin receptor β. Overall, the cumulative risk of diabetes post-COVID-19 is closely tied to age, suggesting more attention should be paid to blood sugar management in elderly COVID-19 patients.

Persistence of SARS-CoV-2 colonization and high expression of inflammatory factors in cardiac tissue 6 months after COVID-19 recovery: a prospective cohort study.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in myocardial autopsy tissues has been observed in certain individuals with coronavirus disease 2019 (COVID-19). However, the duration of cardiac involvement remains uncertain among recovered COVID-19 patients. Our study aims to evaluate the long-term persistence of SARS-CoV-2 within cardiac tissue. We prospectively and consecutively evaluated the patients undergoing mitral valve replacement (MVR) and left atrial (LA) volume reduction surgery from May 25 to June 10, 2023 at our center, who had been approximately 6 months of recovery after Omicron wave. Patients tested positive for SARS-CoV-2 upon admission were excluded. The surgical LA tissue was collected in RNA preservation solution and stored at -80 ℃ immediately. Then SARS-CoV-2, interleukin-6 (IL-6) and interleukin-1β (IL-1β) RNA expression in LA tissues were assessed through thrice-repeated reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. Categorical variables were assessed using the Chi-square or Fisher's exact tests, and continuous variables was analyzed using the Mann-Whitney U test. Nine of 41 patients were enrolled, all of whom tested negative for SARS-CoV-2 upon admission (two antigen and PCR tests). In four of nine patients, SARS-CoV-2 RNA was detected in their LA tissue, indicating viral colonization. Among the four positive cases, the IL-6 and IL-1β relative expression levels in the LA tissue of one patient were increased approximately 55- and 110-fold, respectively, compared to those of SARS-CoV-2 (-) patients. Increased expression of IL-6 and IL-1β were observed in the myocardium of this patient. Another patient demonstrated a remarkable 7-fold increase in both IL-6 and IL-1β expression, surpassing that of SARS-CoV-2 (-) patients. Additionally, no other cardiac inflammation-related diseases or conditions were presented in these two patients. The IL-6 and IL-1β expression levels of the remaining two patients were not significantly different from those of SARS-CoV-2 (-) patients. The relative expression levels of IL-6 and IL-1β in cardiac tissues of all SARS-CoV-2 (-) patients were relatively low. Interestingly, despite abnormally elevated levels of IL-6 and IL-1β within their cardiac tissue, two patients did not show a significant increase in serum IL-6 and IL-1β levels when compared to other patients. Our research suggests that certain COVID-19-recovered patients have persistent colonization of SARS-CoV-2 in their cardiac tissue, accompanied by a local increase in inflammatory factors.

Noninvasive Autopsy-Validated Tumor Probability Maps Identify Glioma Invasion Beyond Contrast Enhancement.

This study identified a clinically significant subset of patients with glioma with tumor outside of contrast enhancement present at autopsy and subsequently developed a method for detecting nonenhancing tumor using radio-pathomic mapping. We tested the hypothesis that autopsy-based radio-pathomic tumor probability maps would be able to noninvasively identify areas of infiltrative tumor beyond traditional imaging signatures. A total of 159 tissue samples from 65 subjects were aligned to MRI acquired nearest to death for this retrospective study. Demographic and survival characteristics for patients with and without tumor beyond the contrast-enhancing margin were computed. An ensemble algorithm was used to predict pixelwise tumor presence from pathological annotations using segmented cellularity (Cell), extracellular fluid, and cytoplasm density as input (6 train/3 test subjects). A second level of ensemble algorithms was used to predict voxelwise Cell, extracellular fluid, and cytoplasm on the full data set (43 train/22 test subjects) using 5-by-5 voxel tiles from T1, T1 + C, fluid-attenuated inversion recovery, and apparent diffusion coefficient as input. The models were then combined to generate noninvasive whole brain maps of tumor probability. Tumor outside of contrast was identified in 41.5% of patients, who showed worse survival outcomes (hazard ratio = 3.90, P < .001). Tumor probability maps reliably tracked nonenhancing tumor on a range of local and external unseen data, identifying tumor outside of contrast in 69% of presurgical cases that also showed reduced survival outcomes (hazard ratio = 1.67, P = .027). This study developed a multistage model for mapping gliomas using autopsy tissue samples as ground truth, which was able to identify regions of tumor beyond traditional imaging signatures.

Virtual histological staining of unlabeled autopsy tissue

Traditional histochemical staining of post-mortem samples often confronts inferior staining quality due to autolysis caused by delayed fixation of cadaver tissue, and such chemical staining procedures covering large tissue areas demand substantial labor, cost and time. Here, we demonstrate virtual staining of autopsy tissue using a trained neural network to rapidly transform autofluorescence images of label-free autopsy tissue sections into brightfield equivalent images, matching hematoxylin and eosin (H&E) stained versions of the same samples. The trained model can effectively accentuate nuclear, cytoplasmic and extracellular features in new autopsy tissue samples that experienced severe autolysis, such as COVID-19 samples never seen before, where the traditional histochemical staining fails to provide consistent staining quality. This virtual autopsy staining technique provides a rapid and resource-efficient solution to generate artifact-free H&E stains despite severe autolysis and cell death, also reducing labor, cost and infrastructure requirements associated with the standard histochemical staining.

Brain banking in the United States and Europe: Importance, challenges, and future trends.

In recent years, brain banks have become valuable resources for examining the molecular underpinnings of various neurological and psychological disorders including Alzheimer disease and Parkinson disease. However, the availability of brain tissue has significantly declined. Proper collection, preparation, and preservation of postmortem autopsy tissue are essential for optimal downstream brain tissue distribution and experimentation. Collaborations between brain banks through larger networks such as NeuroBioBank with centralized sample request mechanisms promote tissue distribution where brain donations are disproportionately lower. Collaborations between brain banking networks also help to standardize the brain donation and sample preparation processes, ensuring proper distribution and experimentation. Ethical brain donation and thorough processing enhances the responsible conduct of scientific studies. Education and outreach programs that foster collaboration between hospitals, nursing homes, neuropathologists, and other research scientists help to alleviate concerns among potential brain donors. Furthermore, ensuring that biorepositories accurately reflect the true demographics of communities will result in research data that reliably represent populations. Implementing these measures will grant scientists improved access to brain tissue, facilitating a deeper understanding of the neurological diseases that impact millions.

Neutrophil Activity and Extracellular Matrix Degradation: Drivers of Lung Tissue Destruction in Fatal COVID-19 Cases and Implications for Long COVID.

Pulmonary fibrosis, severe alveolitis, and the inability to restore alveolar epithelial architecture are primary causes of respiratory failure in fatal COVID-19 cases. However, the factors contributing to abnormal fibrosis in critically ill COVID-19 patients remain unclear. This study analyzed the histopathology of lung specimens from eight COVID-19 and six non-COVID-19 postmortems. We assessed the distribution and changes in extracellular matrix (ECM) proteins, including elastin and collagen, in lung alveoli through morphometric analyses. Our findings reveal the significant degradation of elastin fibers along the thin alveolar walls of the lung parenchyma, a process that precedes the onset of interstitial collagen deposition and widespread intra-alveolar fibrosis. Lungs with collapsed alveoli and organized fibrotic regions showed extensive fragmentation of elastin fibers, accompanied by alveolar epithelial cell death. Immunoblotting of lung autopsy tissue extracts confirmed elastin degradation. Importantly, we found that the loss of elastin was strongly correlated with the induction of neutrophil elastase (NE), a potent protease that degrades ECM. This study affirms the critical role of neutrophils and neutrophil enzymes in the pathogenesis of COVID-19. Consistently, we observed increased staining for peptidyl arginine deiminase, a marker for neutrophil extracellular trap release, and myeloperoxidase, an enzyme-generating reactive oxygen radical, indicating active neutrophil involvement in lung pathology. These findings place neutrophils and elastin degradation at the center of impaired alveolar function and argue that elastolysis and alveolitis trigger abnormal ECM repair and fibrosis in fatal COVID-19 cases. Importantly, this study has implications for severe COVID-19 complications, including long COVID and other chronic inflammatory and fibrotic disorders.

Multiomic Analysis of Neuroinflammation and Occult Infection in Sudden Infant Death Syndrome

Antemortem infection is a risk factor for sudden infant death syndrome (SIDS)-the leading postneonatal cause of infant mortality in the developed world. Manifestations of infection and inflammation are not always apparent in clinical settings or by standard autopsy; thus, enhanced resolution approaches are needed. To ascertain whether a subset of SIDS cases is associated with neuroinflammation and occult infection. In this case-control study, postmortem fluids from SIDS cases and controls collected between July 2011 and November 2018 were screened for elevated inflammatory markers, specifically cerebrospinal fluid (CSF) neopterin and CSF and serum cytokines. CSF, liver, and brain tissue from SIDS cases with elevated CSF neopterin were subjected to metagenomic next-generation sequencing (mNGS) to probe for infectious pathogens. Brainstem tissue from a subset of these cases was analyzed by single-nucleus RNA sequencing (snRNAseq) to measure cell type-specific gene expression associated with neuroinflammation and infection. All tissue and fluid analyses were performed from April 2019 to January 2023 in a pathology research laboratory. Included was autopsy material from infants dying of SIDS and age-matched controls dying of known causes. There were no interventions or exposures. CSF neopterin levels were measured by high-performance liquid chromatography. Cytokines were measured by multiplex fluorometric assay. mNGS was performed on liver, CSF, brain, and brainstem tissue. snRNAseq was performed on brainstem tissue. A cohort of 71 SIDS cases (mean [SD] age, 55.2 [11.4] postconceptional weeks; 42 male [59.2%]) and 20 controls (mean [SD] age, 63.2 [16.9] postconceptional weeks; 11 male [55.0%]) had CSF and/or serum available. CSF neopterin was screened in 64 SIDS cases and 15 controls, with no exclusions. Tissues from 6 SIDS cases were further analyzed. For CSF neopterin measures, SIDS samples were from infants with mean (SD) age of 54.5 (11.3) postconceptional weeks (38 male [59.4%]) and control samples were from infants with mean (SD) age of 61.5 (17.4) postconceptional weeks (7 male [46.7%]). A total of 6 SIDS cases (9.3%) with high CSF neopterin were identified, suggestive of neuroinflammation. mNGS detected human parechovirus 3 (HPeV3) in tissue and CSF from 1 of these 6 cases. snRNAseq of HPeV3-positive brainstem tissue (medulla) revealed dramatic enrichment of transcripts for genes with predominately inflammatory functions compared with 3 age-matched SIDS cases with normal CSF neopterin levels. Next-generation molecular tools in autopsy tissue provide novel insight into pathogens that go unrecognized by normal autopsy methodology, including in infants dying suddenly and unexpectedly.

Pathology and Monkeypox virus Localization in Tissues From Immunocompromised Patients With Severe or Fatal Mpox.

Pathology and Monkeypox virus (MPXV) tissue tropism in severe and fatal human mpox is not thoroughly described but can help elucidate the disease pathogenesis and the role of coinfections in immunocompromised patients. We analyzed biopsy and autopsy tissues from 22 patients with severe or fatal outcomes to characterize pathology and viral antigen and DNA distribution in tissues by immunohistochemistry and in situ hybridization. Tissue-based testing for coinfections was also performed. Mucocutaneous lesions showed necrotizing and proliferative epithelial changes. Deceased patients with autopsy tissues evaluated had digestive tract lesions, and half had systemic tissue necrosis with thrombotic vasculopathy in lymphoid tissues, lung, or other solid organs. Half also had bronchopneumonia, and one-third had acute lung injury. All cases had MPXV antigen and DNA detected in tissues. Coinfections were identified in 5 of 16 (31%) biopsy and 4 of 6 (67%) autopsy cases. Severe mpox in immunocompromised patients is characterized by extensive viral infection of tissues and viremic dissemination that can progress despite available therapeutics. Digestive tract and lung involvement are common and associated with prominent histopathological and clinical manifestations. Coinfections may complicate mpox diagnosis and treatment. Significant viral DNA (likely correlating to infectious virus) in tissues necessitates enhanced biosafety measures in healthcare and autopsy settings.

The changes in tissue histomorphology and quality of DNA in healthy human autopsied tissues stored at −20 °C and −150 °C

The aim of this study was to compare the changes in tissue histomorphology and DNA quality in six different healthy tissues (brain, heart, lung, liver, spleen and kidney) exempted during autopsy of healthy individuals and storage at −20 °C and −150 °C three month. Tissue samples were obtained, divided by tissue and temperature group, and for each sample, tissue histomorphology and DNA (isolated from all tissues in duplicated − 72 samples of DNA) quality were analysed. Morphology of tissue samples was studied using H&E staining. DNA was isolated using the phenol–chloroform-isoamyl alcohol method. To assess the concentration and purity of the DNA samples, we used a spectrophotometer to measure absorbance at wavelengths of 280 nm and 260 nm. The fragments of human telomerase reverse transcriptase (hTERT) gene were amplified from the DNA using PCR reaction and then visualised using the 2 % agarose gel. Samples stored at −150 °C sustained the highest degree of histomorphological damage, while samples stored at −20 °C were less degraded, compared to control. The liver samples stored at −20 °C had a mean DNA concentration (1030.4 ± 51.5 ng/μl) higher than the samples of liver tissue stored at −150 °C (497.4 ± 167.1 ng/μl) (p < 0.001). Other tissues did not have statistically significantly different DNA concentration at both temperatures. Liver samples at −20 °C had degraded DNA, showed as the absence of hTERT gene in most of samples. Other tissue samples in both temperature groups had unfragmented DNA. Storing tissue samples at −20 °C is not inferior in terms of DNA yield and integrity, and possibly superior for tissue histomorphology, comparing with samples stored at −150⁰C.

Viral uptake and pathophysiology of the lung endothelial cells in age-associated severe SARS-CoV-2 infection models.

Thrombosis is the major cause of death in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and the pathology of vascular endothelial cells (ECs) has received much attention. Although there is evidence of the infection of ECs in human autopsy tissues, their detailed pathophysiology remains unclear due to the lack of animal model to study it. We used a mouse-adapted SARS-CoV-2 virus strain in young and mid-aged mice. Only mid-aged mice developed fatal pneumonia with thrombosis. Pulmonary ECs were isolated from these infected mice and RNA-Seq was performed. The pulmonary EC transcriptome revealed that significantly higher levels of viral genes were detected in ECs from mid-aged mice with upregulation of viral response genes such as DDX58 and IRF7. In addition, the thrombogenesis-related genes encoding PLAT, PF4, F3 PAI-1, and P-selectin were upregulated. In addition, the inflammation-related molecules such as CXCL2 and CXCL10 were upregulated in the mid-aged ECs upon viral infection. Our mouse model demonstrated that SARS-CoV-2 virus entry into aged vascular ECs upregulated thrombogenesis and inflammation-related genes and led to fatal pneumonia with thrombosis. Current results of EC transcriptome showed that EC uptake virus and become thrombogenic by activating neutrophils and platelets in the aged mice, suggesting age-associated EC response as a novel finding in human severe COVID-19.