Histopathological evaluation of abdominal aortic aneurysms with deep learning.

Computational analysis of histopathological specimens holds promise in identifying biomarkers, elucidating disease mechanisms, and streamlining clinical diagnosis. However, the application of deep learning techniques in vascular pathology remains underexplored. Here, we present a comprehensive evaluation of deep learning-based approaches to analyze digital whole-slide images of abdominal aortic aneurysm samples from 369 patients from three European centers. Deep learning demonstrated robust performance in predicting inflammatory characteristics, particularly in the adventitia, as well as fibrosis grade and remaining elastic fibers in the tunica media. Overall, this study represents the first comprehensive evaluation of computational pathology in vascular disease and has the potential to contribute to improved understanding of abdominal aortic aneurysm pathophysiology and personalization of treatment strategies, particularly when integrated with radiological phenotypes and clinical outcomes.

Proteomic Atlas of Atherosclerosis: The Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes, and Outcomes.

BACKGROUND: Using proteomics, we aimed to reveal molecular types of human atherosclerotic lesions and study their associations with histology, imaging, and cardiovascular outcomes. METHODS: Two hundred nineteen carotid endarterectomy samples were procured from 120 patients. A sequential protein extraction protocol was employed in conjunction with multiplexed, discovery proteomics. To focus on extracellular proteins, parallel reaction monitoring was employed for targeted proteomics. Proteomic signatures were integrated with bulk, single-cell, and spatial RNA-sequencing data, and validated in 200 patients from the Athero-Express Biobank study. RESULTS: This extensive proteomics analysis identified plaque inflammation and calcification signatures, which were inversely correlated and validated using targeted proteomics. The inflammation signature was characterized by the presence of neutrophil-derived proteins, such as S100A8/9 (calprotectin) and myeloperoxidase, whereas the calcification signature included fetuin-A, osteopontin, and gamma-carboxylated proteins. The proteomics data also revealed sex differences in atherosclerosis, with large-aggregating proteoglycans versican and aggrecan being more abundant in females and exhibiting an inverse correlation with estradiol levels. The integration of RNA-sequencing data attributed the inflammation signature predominantly to neutrophils and macrophages, and the calcification and sex signatures to smooth muscle cells, except for certain plasma proteins that were not expressed but retained in plaques, such as fetuin-A. Dimensionality reduction and machine learning techniques were applied to identify 4 distinct plaque phenotypes based on proteomics data. A protein signature of 4 key proteins (calponin, protein C, serpin H1, and versican) predicted future cardiovascular mortality with an area under the curve of 75% and 67.5% in the discovery and validation cohort, respectively, surpassing the prognostic performance of imaging and histology. CONCLUSIONS: Plaque proteomics redefined clinically relevant patient groups with distinct outcomes, identifying subgroups of male and female patients with elevated risk of future cardiovascular events.

Completing the view - histologic insights from circular AAA specimen including 3D imaging : A methodologic approach towards histologic analysis of circumferential AAA samples.

Abdominal aortic aneurysm (AAA) is a pathologic enlargement of the infrarenal aorta with an associated risk of rupture. However, the responsible mechanisms are only partially understood. Based on murine and human samples, a heterogeneous distribution of characteristic pathologic features across the aneurysm circumference is expected. Yet, complete histologic workup of the aneurysm sac is scarcely reported. Here, samples from five AAAs covering the complete circumference partially as aortic rings are investigated by histologic means (HE, EvG, immunohistochemistry) and a new method embedding the complete ring. Additionally, two different methods of serial histologic section alignment are applied to create a 3D view. The typical histopathologic features of AAA, elastic fiber degradation, matrix remodeling with collagen deposition, calcification, inflammatory cell infiltration and thrombus coverage were distributed without recognizable pattern across the aneurysm sac in all five patients. Analysis of digitally scanned entire aortic rings facilitates the visualization of these observations. Immunohistochemistry is feasible in such specimen, however, tricky due to tissue disintegration. 3D image stacks were created using open-source and non-generic software correcting for non-rigid warping between consecutive sections. Secondly, 3D image viewers allowed visualization of in-depth changes of the investigated pathologic hallmarks. In conclusion, this exploratory descriptive study demonstrates a heterogeneous histomorphology around the AAA circumference. Warranting an increased sample size, these results might need to be considered in future mechanistic research, especially in reference to intraluminal thrombus coverage. 3D histology of such circular specimen could be a valuable visualization tool for further analysis.

The Incidence of IgG4-Related and Inflammatory Abdominal Aortic Aneurysm Is Rare in a 101 Patient Cohort.

Abdominal aortic aneurysms (AAA) are the most frequent aortic dilation, with considerable morbidity and mortality. Inflammatory (infl) and IgG4-positive AAAs represent specific subtypes of unclear incidence and clinical significance. Here, histologic and serologic analyses with retrospective clinical data acquisition are investigated via detailed histology, including morphologic (HE, EvG: inflammatory subtype, angiogenesis, and fibrosis) and immunhistochemic analyses (IgG and IgG4). In addition, complement factors C3/C4 and immunoglobulins IgG, IgG2, IgG4 and IgE were measured in serum samples and clinical data uses patients' metrics, as well as through semi-automated morphometric analysis (diameter, volume, angulation and vessel tortuosity). A total of 101 eligible patients showed five (5%) IgG4 positive (all scored 1) and seven (7%) inflammatory AAAs. An increased degree of inflammation was seen in IgG4 positive and inflAAA, respectively. However, serologic analysis revealed no increased levels of IgG or IgG4. The operative procedure time was not different for those cases and the short-term clinical outcomes were equal for the entire AAA cohort. Overall, the incidence of inflammatory and IgG4-positive AAA samples seems very low based on histologic and serum analyses. Both entities must be considered distinct disease phenotypes. Short-term operative outcomes were not different for both sub-cohorts.

Soluble ST2 in Patients with Carotid Artery Stenosis-Association with Plaque Morphology and Long-Term Outcome.

Interleukin (IL-33) and the ST2 receptor are implicated in the pathogenesis of atherosclerosis. Soluble ST2 (sST2), which negatively regulates IL-33 signaling, is an established biomarker in coronary artery disease and heart failure. Here we aimed to investigate the association of sST2 with carotid atherosclerotic plaque morphology, symptom presentation, and the prognostic value of sST2 in patients undergoing carotid endarterectomy. A total of 170 consecutive patients with high-grade asymptomatic or symptomatic carotid artery stenosis undergoing carotid endarterectomy were included in the study. The patients were followed up for 10 years, and the primary endpoint was defined as a composite of adverse cardiovascular events and cardiovascular mortality, with all-cause mortality as the secondary endpoint. The baseline sST2 showed no association with carotid plaque morphology assessed using carotid duplex ultrasound (B 0.051, 95% CI -0.145-0.248, p = 0.609), nor with modified histological AHA classification based on morphological description following surgery (B -0.032, 95% CI -0.194-0.130, p = 0.698). Furthermore, sST2 was not associated with baseline clinical symptoms (B -0.105, 95% CI -0.432-0.214, p = 0.517). On the other hand, sST2 was an independent predictor for long-term adverse cardiovascular events after adjustment for age, sex, and coronary artery disease (HR 1.4, 95% CI 1.0-2.4, p = 0.048), but not for all-cause mortality (HR 1.2, 95% CI 0.8-1.7, p = 0.301). Patients with high baseline sST2 levels had a significantly higher adverse cardiovascular event rate as compared to patients with lower sST2 (log-rank p < 0.001). Although IL-33 and ST2 play a role in the pathogenesis of atherosclerosis, sST2 is not associated with carotid plaque morphology. However, sST2 is an excellent prognostic marker for long-term adverse cardiovascular outcomes in patients with high-grade carotid artery stenosis.

Multifactorial White Matter Damage in the Acute Phase and Pre-Existing Conditions May Drive Cognitive Dysfunction after SARS-CoV-2 Infection: Neuropathology-Based Evidence.

BACKGROUND: There is an urgent need to better understand the mechanisms underlying acute and long-term neurological symptoms after COVID-19. Neuropathological studies can contribute to a better understanding of some of these mechanisms. METHODS: We conducted a detailed postmortem neuropathological analysis of 32 patients who died due to COVID-19 during 2020 and 2021 in Austria. RESULTS: All cases showed diffuse white matter damage with a diffuse microglial activation of a variable severity, including one case of hemorrhagic leukoencephalopathy. Some cases revealed mild inflammatory changes, including olfactory neuritis (25%), nodular brainstem encephalitis (31%), and cranial nerve neuritis (6%), which were similar to those observed in non-COVID-19 severely ill patients. One previously immunosuppressed patient developed acute herpes simplex encephalitis. Acute vascular pathologies (acute infarcts 22%, vascular thrombosis 12%, diffuse hypoxic-ischemic brain damage 40%) and pre-existing small vessel diseases (34%) were frequent findings. Moreover, silent neurodegenerative pathologies in elderly persons were common (AD neuropathologic changes 32%, age-related neuronal and glial tau pathologies 22%, Lewy bodies 9%, argyrophilic grain disease 12.5%, TDP43 pathology 6%). CONCLUSIONS: Our results support some previous neuropathological findings of apparently multifactorial and most likely indirect brain damage in the context of SARS-CoV-2 infection rather than virus-specific damage, and they are in line with the recent experimental data on SARS-CoV-2-related diffuse white matter damage, microglial activation, and cytokine release.

Elevated EMMPRIN Serum Levels Indicate Plaque Vulnerability in Patients With Asymptomatic High Grade Carotid Stenosis.

OBJECTIVE: Carotid atherosclerosis is an important cause of cerebral ischaemic stroke. Sonographic plaque characteristics are inappropriate for exact prediction of possible future ischaemic events. Additional markers are needed to predict the clinical outcome in high grade carotid stenosis. This study aimed to test extracellular matrix metalloproteinase inducer (EMMPRIN), due to its involvement in plaque formation and destabilisation, as a potential marker of high risk vulnerable plaques. METHODS: EMMPRIN was analysed in pre-operative serum samples from patients with symptomatic and asymptomatic carotid artery stenosis by a specific ELISA. Pre-operative duplex sonography classified the atherosclerotic plaque due to echogenicity. Histopathological analysis of vulnerable and non-vulnerable plaques was based on the American Heart Association (AHA) classification. RESULTS: The study included 265 patients undergoing carotid endarterectomy: 90 (m:f, 69:21) patients with symptomatic and 175 (m:f, 118:57) with asymptomatic disease. Analysis of circulating EMMPRIN revealed significantly higher levels in patients with echolucent plaques (4 480; IQR 3 745, 6 144 pg/mL) compared with echogenic plaques (4 159; IQR 3 418, 5 402 pg/mL; p = .025). Asymptomatic patients with vulnerable plaques had significantly higher levels of EMMPRIN (4 875; IQR 3 850, 7 016 pg/mL) compared with non-vulnerable plaques (4 109; IQR 3 433, 5 402 pg/mL; p < .001). In logistic regression analysis, duplex sonography combined with age, gender, and clinical risk factors predicted vulnerable plaques in asymptomatic patients with an AUC of 0.71 (95% CI 0.61 - 0.80). EMMPRIN significantly improved the AUC in asymptomatic patients (AUC 0.79; 95% CI 0.71 - 0.87; p = .014). CONCLUSION: Patients with high risk plaques according to ultrasound and histopathological characteristics demonstrated increased serum EMMPRIN levels. EMMPRIN on top of clinical risk factors, including age, gender, and duplex sonography may be used for pre-operative risk stratification in asymptomatic patients.

Ex vivo fluorescence confocal microscopy: chances and changes in the analysis of breast tissue.

BACKGROUND: Rapid histologic diagnosis of frozen sections is essential for a variety of surgical procedures. Frozen sections however, require specialized lab equipment, are prone to freezing artifacts and are not applicable to all types of tissue. Adipose tissue is especially difficult to process in frozen sections. Although these limitations are well known, no alternative method for microscopic tissue analysis that might replace frozen sections could be established. Our objective was to evaluate whether tissue imaging based on ex vivo fluorescent confocal microscopy (FCM) is applicable for rapid microscopic assessment of breast tumors specimens with abundant adipose tissue. METHODS: We evaluated 17 tissue samples from mastectomy specimens, rich in adipose tissue, submitted to the department of pathology at the Medical University of Vienna. We conducted our study on the FCM VivaScope® 2500M-G4 (Mavig GmbH, Munich, Germany; Caliber I.D.; Rochester NY, USA). RESULTS: When comparing FCM to frozen sections, we found a very similar overall processing time for FCM images and frozen sections respectively. Image quality was mostly superior to frozen sections (especially for adipose tissue and nuclear detail) but inferior to H&E stained FFPE sections. Limitations of the technology were uneven coloring, invisibility of ink applied for marking tissue margins and distortion artifacts if too much pressure is applied to the tissue. CONCLUSION: FCM has the potential to expand the application and usefulness of rapid tissue analysis as speed is comparable and quality exceeds that of frozen sections especially in tissues rich in adipose cells such as breast specimen.

Computer-assisted evaluation enhances the quantification of interstitial fibrosis in renal implantation biopsies, measures differences between frozen and paraffin sections, and predicts delayed graft function.

BACKGROUND: (Pre-)Implantation biopsies provide important data on the quality of donor kidneys. Interstitial fibrosis, as a known predictor for kidney disease progression, is an essential feature of this evaluation. However, the assessment of frozen sections of implantation biopsies is challenging and can result in the disposal of candidate organs. We sought to apply digital image analysis (DIA) to quantify the differences between frozen and paraffin sections when evaluating interstitial fibrosis, identify factors that influence these variations and test the predictive value of the computerised measures. METHODS: We quantified the differences between frozen and paraffin sections in the same biopsy samples by measuring Sirius red-stained interstitial areas (SRIA) in DIA. We compared them to the original reports, and retrospectively correlated our findings to clinical data, graft function and outcome in 73 patients. RESULTS: Frozen sections display a broader interstitial area than paraffin sections, in some cases up to one-third more (mean difference + 7.8%, range - 7 to 29%). No donor-related factors (age or gender, cold ischemia time, or non-heart-beating donor) influenced significantly this difference. Compared to the original assessment of frozen vs paraffin sections in optical microscopy, the DIA of interstitial fibrosis shows a higher consistency (ICC 0.69). Our approach further allows to distinguish SRIA in paraffin sections as an independent predictor for delayed graft function (OR = 1.1; p = 0.028). CONCLUSIONS: DIA is superior to and more consistent than routine optic microscopy for interstitial fibrosis evaluation. This method could improve implantation biopsy diagnostics and help to reduce disposal of organs.

DNA methylation profiles differ in responders versus non-responders to anti-PD-1 immune checkpoint inhibitors in patients with advanced and metastatic head and neck squamous cell carcinoma.

BACKGROUND: Biomarkers for response prediction to anti-programmed cell death 1 (PD-1) immune checkpoint inhibitors (ICI) in patients with head and neck squamous cell carcinoma (HNSCC) are urgently needed for a personalized therapy approach. We investigated the predictive potential of inflammatory parameters and DNA methylation profiling in patients with HNSCC treated with anti-PD-1 ICI. METHODS: We identified patients with HNSCC that were treated with anti-PD-1 ICI therapy in the recurrent or metastatic setting after progression to platinum-based chemotherapy in two independent centers. We analyzed DNA methylation profiles of >850.000 CpG sites in tumor specimens of these patients by Infinium MethylationEPIC microarrays, immune cell density in the tumor microenvironment (CD8, CD3, CD45RO, forkhead box P3 (FOXP3), CD68), PD-1 and programmed cell death ligand 1 (PD-L1) expression by immunohistochemistry, and blood inflammation markers (platelet-to-lymphocyte ratio, leucocyte-to-lymphocyte ratio, monocyte-to-lymphocyte ratio, neutrophil-to-lymphocyte ratio). DNA methylation profiles and immunological markers were bioinformatically and statistically correlated with radiological response to anti-PD-1 ICI. RESULTS: 37 patients with HNSCC (median age of 62 years; range 49-83; 8 (21.6%) women, 29 (78.4%) men) were included (Center 1 N=26, 70.3%; Center 2 N=11, 29.7%). Median number of prior systemic therapies was 1 (range 1-4). Five out of 37 (13.5%) patients achieved an objective response to ICI. Median progression-free survival and median overall survival times were 3.7 months (range 0-22.9) and 9.0 months (range 0-38.8), respectively. Microarray analyses revealed a methylation signature including both hypomethylation and hypermethylation which was predictive for response to ICI and included several genes involved in cancer-related molecular pathways. Over-represented differentially methylated genes between responders and non-responders were associated with 'Axon guidance', 'Hippo signaling', 'Pathways in cancer' and 'MAPK signaling'. A statistically significant correlation of PD-L1 expression and response was present (p=0.0498). CONCLUSIONS: Our findings suggest that tumor DNA methylation profiling may be useful to predict response to ICI in patients with HNSCC.

Torque Teno Virus Load Is Associated With Subclinical Alloreactivity in Kidney Transplant Recipients: A Prospective Observational Trial.

BACKGROUND: Nonpathogenic torque teno viruses (TTVs) are highly prevalent in transplant recipients and associated with immunosuppression. Studies in kidney transplant patients have proposed assessment of TTV load for risk stratification of clinically overt graft rejection. The value of TTV quantification in the context of subclinical rejection has not been evaluated. METHODS: In this prospective trial, 307 consecutive kidney transplant recipients were subjected to per-protocol monitoring of plasma TTV. TTV was analyzed in the context of protocol biopsies (n = 82), scheduled 1 year posttransplantation. RESULTS: TTV load at the time of biopsy was lower in recipients with rejection (n = 19; according to Banff, including borderline changes suspicious for acute T cell-mediated rejection) than those without rejection (n = 63) whereby each log increase in TTV copies/mL decreased the risk for rejection by 9% (risk ratio 0.91, 95% confidence interval, 0.85-0.97; P = 0.004). Development of chronic lesions (cg, cv, ci, ct, ah, ptcml) was associated with the number of days with a TTV load <1 × 106 copies/mL between months 3 and 12 posttransplant (ß 0.07, 95% confidence interval, 0.01-0.14; P = 0.02). CONCLUSIONS: This trial demonstrates an association between TTV and subclinical graft rejection in kidney transplant recipients. A TTV load <1 × 106 copies/mL suggests suboptimal immunosuppression.

Magnetic Resonance Imaging for Evaluation of Interstitial Fibrosis in Kidney Allografts.

Interstitial fibrosis (IF) is the common pathway of chronic kidney injury in various conditions. Magnetic resonance imaging (MRI) may be a promising tool for the noninvasive assessment of IF in renal allografts. METHODS: This prospective trial was primarily designed to investigate whether the results of T1-weighted MRI associate with the degree of IF. Thirty-two kidney transplant recipients were subjected to 1.5-Tesla MRI scans shortly before or after routine allograft biopsies. MRI parameters [T1 and T2 relaxation times; apparent diffusion coefficient (ADC)] were assessed for cortical and medullary sections. RESULTS: Advanced IF (Banff ci score >1) was associated with higher cortical T1 (but not T2) values [1451 (median; interquartile range: 1331-1506) versus 1306 (1197-1321) ms in subjects with ci scores ≤1; P = 0.011; receiver operating characteristic area under the curve for prediction of ci > 1: 0.76]. In parallel, T1 values were associated with kidney function and proteinuria. There was also a relationship between IF and corticomedullary differences on ADC maps (receiver operating characteristic area under the curve for prediction of ci ≤ 1: 0.79). CONCLUSIONS: Our results support the use of MRI for noninvasive assessment of allograft scarring. Future studies will have to clarify the role of T1 (and ADC) mapping as a surrogate endpoint reflecting the progression of chronic graft damage.