Flanagan's condensed protocol for neurodegenerative diseases. Implementation in a clinical autopsy setting with partial supervision of a neuropathologist.

The Condensed Protocol (CP) was originally developed for the evaluation of Alzheimer's Disease (AD) and other neurodegenerative diseases as a workable alternative to the complex and costly established autopsy guidelines. The study objective is to examine the degree of implementation of the CP in the pathology department of a third level university hospital in a period of 5 years. Clinical autopsies performed between 2016 and 2021 on patients aged 65 years or over and did not require a specific neuropathological examination were reviewed. Histological screening and staging of neurodegenerative diseases was performed using the original immunohistochemical stains. Out of 255 autopsies, 204 met the inclusion criteria and 190 could be reviewed. The CP was applied to 99 cases; histological signs of neurodegenerative disease were observed in 92. Sampling errors were detected in 59 cases. Immunohistochemical studies were performed in 68 cases. The diseases identified were: 31 cases of AD (12 low grade; 19 intermediate), 18 amyloid angiopathy, 15 primary age-related tauopathy, 6 argyrophilic grain disease, 3 progressive supranuclear palsy, 1 Lewy body disease (of 22 cases), and 2 limbic-predominant age TDP43 encephalopathy (of 5 cases). In 30 out of 83 cases, there was more severe vascular pathology in complete sections of frontal cortex and lentiform nucleus. The CP allows reliable detection and staging of AD and related neurodegenerative diseases in clinical autopsies. However, supervision by a neuropathologist seems necessary for a fully successful implementation of the CP in a clinical hospital setting.

Gal-1 Expression Analysis in the GLIOCAT Multicenter Study: Role as a Prognostic Factor and an Immune-Suppressive Biomarker.

Glioblastoma (GBM) is the most frequent primary malignant brain tumor and has a dismal prognosis. Unfortunately, despite the recent revolution of immune checkpoint inhibitors in many solid tumors, these have not shown a benefit in overall survival in GBM patients. Therefore, new potential treatment targets as well as diagnostic, prognostic, and/or predictive biomarkers are needed to improve outcomes in this population. The ß-galactoside binding protein Galectin-1 (Gal-1) is a protein with a wide range of pro-tumor functions such as proliferation, invasion, angiogenesis, and immune suppression. Here, we evaluated Gal-1 expression by immunohistochemistry in a homogenously treated cohort of GBM (the GLIOCAT project) and correlated its expression with clinical and molecular data. We observed that Gal-1 is a negative prognostic factor in GBM. Interestingly, we observed higher levels of Gal-1 expression in the mesenchymal/classical subtypes compared to the less aggressive proneural subtype. We also observed a Gal-1 expression correlation with immune suppressive signatures of CD4 T-cells and macrophages, as well as with several GBM established biomarkers, including SHC1, PD-L1, PAX2, MEOX2, YKL-40, TCIRG1, YWHAG, OLIG2, SOX2, Ki-67, and SOX11. Moreover, Gal-1 levels were significantly lower in grade 4 IDH-1 mutant astrocytomas, which have a better prognosis. Our results confirm the role of Gal-1 as a prognostic factor and also suggest its value as an immune-suppressive biomarker in GBM.

Expression of Protein SOX9 in Biliary Atresia.

OBJECTIVES: Biliary atresia (BA) is still an enigmatic disease. Deeper knowledge of its pathophysiology could help develop better treatments. SOX9 regulates bile duct development, liver regeneration and fibrosis; therefore, it could be determinant in characterizing BA liver damage. Aim: To study if there is a SOX9 expression pattern in liver biopsies from BA patients. METHODS: Liver biopsies from BA patients (group BA), from age-matched infants without primary hepatic disease (group Control), and from patients with other liver conditions (group OLC) were compared. Expression of SOX9 was checked for: amount, intensity of immunoreaction, localization within ductular structures, perifibrotic epithelial cells, and lobular cells. The scores were added to create a scale from 0 to 11 that allowed group comparison. SOX9 Scale and liver survival were also looked for a correlation. RESULTS: All BA cases had a score >4, while all controls scored <4. OLC livers scored 1 to 8 (3.5 ±â€Š2.0) (P < 0.001 between all groups). A cut-off at 4 had 100% sensitivity and 88.24% specificity to differentiate BA from Controls and from OLC (area under receiver operating characteristic curve: 0.9989 (95% confidence interval: 0.9964-1.000). Strong expression of SOX9 was observed mainly in the nuclei of proliferated ductules of portal spaces and fibrotic bridges. SOX9 Scale score could not be related to liver survival in this study. CONCLUSION: In BA livers, SOX9 is mainly expressed in reactive ductular epithelium, following a pattern significantly different from that seen in non-BA patients; thus, SOX9 Scale may have a role in the diagnosis of BA.

Post-mortem neuropathologic examination of a 6-case series of CAR T-cell treated patients.

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. Adverse events are common, and neurotoxicity is one of the most important. However, the physiopathology is unknown and neuropathologic information is scarce. Materials and methods: Post-mortem examination of 6 brains from patients that underwent CAR T-cell therapy from 2017 to 2022. In all cases, polymerase chain reaction (PCR) in paraffin blocks for the detection of CAR T cells was performed. Results: Two patients died of hematologic progression, while the others died of cytokine release syndrome, lung infection, encephalomyelitis, and acute liver failure. Two out of 6 presented neurological symptoms, one with extracranial malignancy progression and the other with encephalomyelitis. The neuropathology of the latter showed severe perivascular and interstitial lymphocytic infiltration, predominantly CD8+, together with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, and a diffuse gliosis of basal ganglia, hippocampus, and brainstem. Microbiological studies were negative for neurotropic viruses, and PCR failed to detect CAR T -cells. Another case without detectable neurological signs showed cortical and subcortical gliosis due to acute hypoxic-ischemic damage. The remaining 4 cases only showed a mild patchy gliosis and microglial activation, and CAR T cells were detected by PCR only in one of them. Conclusions: In this series of patients that died after CAR T-cell therapy, we predominantly found non-specific or minimal neuropathological changes. CAR T-cell related toxicity may not be the only cause of neurological symptoms, and the autopsy could detect additional pathological findings.

Glioblastoma TCGA Mesenchymal and IGS 23 Tumors are Identifiable by IHC and have an Immune-phenotype Indicating a Potential Benefit from Immunotherapy.

PURPOSE: Molecular subtype classifications in glioblastoma may detect therapy sensitivities. IHC would potentially allow the identification of molecular subtypes in routine clinical practice. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tumor samples of 124 uniformly treated, newly diagnosed patients with glioblastoma were submitted to RNA sequencing, IHC, and immune-phenotyping to identify differences in molecular subtypes associated with treatment sensitivities. RESULTS: We detected high molecular and IHC overlapping of the The Cancer Genome Atlas (TCGA) mesenchymal subtype with instrinsic glioma subtypes (IGS) cluster 23 and of the TCGA classical subtype with IGS cluster 18. IHC patterns, gene fusion profiles, and immune-phenotypes varied across subtypes. IHC revealed that the TCGA classical subtype was identified by high expression of EGFR and low expression of PTEN, while the mesenchymal subtype was identified by low expression of SOX2 and high expression of two antibodies, SHC1 and TCIRG1, selected on the basis of RNA differential transcriptomic expression. The proneural subtype was identified by frequent positive IDH1 expression and high Olig2 and Ki67 expression. Immune-phenotyping showed that mesenchymal and IGS 23 tumors exhibited a higher positive effector cell score, a higher negative suppressor cell score, and lower levels of immune checkpoint molecules. The cell-type deconvolution analysis revealed that these tumors are highly enriched in M2 macrophages, resting memory CD4+ T cells, and activated dendritic cells, indicating that they may be ideal candidates for immunotherapy, especially with anti-M2 and/or dendritic cell vaccination. CONCLUSIONS: There is a subset of tumors, frequently classified as mesenchymal or IGS cluster 23, that may be identified with IHC and could well be optimal candidates for immunotherapy.

Midline Cervical Cleft: An Anatomical Finding and a Proposal for a New Approach.

Congenital midline cervical cleft is a rare malformation. Typical case shows an area of hypotrophic skin, a cranial nipple-like structure, and a caudal blind sinus. Cervical extension is limited. Relapse of the retraction is common following cutaneous z-plasty. The aim of this study is to describe the radiological, surgical, and histological findings of the 4 cases treated in our center in the last 8 years and communicate the finding of a contractile structure, anterior to the platysma, composed by striated muscle, figure not previously described. This distinct muscular band is responsible for neck retraction. Removal of this releases cervical tension and is essential to avoid the relapse.

EORTC SPECTA-AYA: A unique molecular profiling platform for adolescents and young adults with cancer in Europe.

For most adolescent and young adult (AYA) cancers, age-specific molecular features are poorly understood. EORTC-SPECTA, an academic translational research infrastructure for biomaterial collection, will explicitly recruit AYA patients and will therefore collect empirical data to bridge the molecular gap between pediatric and adult oncology. The initial pilot study, activated in February 2019 across Europe, will recruit 100 AYA patients (aged 12-29 years) with newly diagnosed or relapsed high-grade gliomas and high-grade bone and soft tissue sarcomas. The primary objective of the pilot is to determine feasibility and recruitment rates. Formalin-fixed tumor tissue and whole blood from study participants will be prospectively collected with clinical data and stored centrally at the Integrated BioBank of Luxembourg. Whole exome sequencing of matched tumor and blood, and tumor RNA sequencing and DNA methylation profiling will be performed at the German Cancer Research Center, Heidelberg, Germany. Virtual central pathology review of scanned diagnostic slides will be undertaken by an international expert panel, and diagnostic material returned to the participating centers. A multidisciplinary molecular tumor board will release a clinically validated report to referring clinicians within 4-6 weeks after biopsy. SPECTA-AYA constitutes a major opportunity to gain knowledge about the tumor biology of this unique age group. It incorporates notable innovative aspects: AYA specificity, pan-European academic collaboration, centralized biobanking, comprehensive molecular profiling and virtual central pathology review, among others. SPECTA-AYA will help untangle the tumor particularities of AYAs with cancer and aims to improve their access to novel drugs and personalized medicine.

Prognostic value of stem cell markers in glioblastoma.

Background/Context: Glioblastoma (GB) is the most common primary brain tumour in adults and it is associated with a high mortality rate. According to the stem cell theory, the growth, relapse and treatment response of GB is determined by the stem cell subpopulation present in the tumour. Objective: Our aim is to study the prognostic value of stem cell markers (CD44, Nestin, Olig2 and SOX2) in a series of homogeneously treated GBs. Material and methods: We study 280 GBs treated with STUPP acheme with a histologican review of the cases and TMA with a máximum of 4 spots for each case. Each slide was immunohistochemically stained and Reading. We compared the immunohistochemical results with survival tme. Results: Only SOX2 immunoexpression (IE) excedding 10% of the tumour cells was found to be related to good survival (p= 0.037) in univariate analysis. However, amultivariate analysis indicate the age, surgery and MGMT promotes methylation but no SOX2 IE are prognostic factors. Conclusions: We conclude the immunohistochemical studies of stem cell markers in GB are not useful for predicting prognosis in daily practice.

GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma.

A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both in vitro and in vivo. Accordingly, a low GPR56-associated signature is prognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappa B (NF-κB) signaling pathway, thereby providing the rationale by which this receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM.

Management in chordoid glioma: Avoiding the pitfalls in this rare and challenging entity.

Chordoid glioma (CG) of the third ventricle is an unusual neoplasm of glial nature, which is almost exclusively located in the anterior wall of the third ventricle, in close relation with the hypothalamus. Magnetic resonance images show CG as a suprasellar, hypo- to isointense mass, homogeneously enhancing after the administration of gadolinium. Since its description in 1998 by Brat et al., approximately 85 cases have been reported. Some of its pathological features are under discussion and its histological origin still remains unclear. In this study, we present a patient having this rare entity. We review the management of CG reported in literature. We also studied its pathological features, the postoperative mortality and morbidity related to radical surgical resection, and the implemented adjuvant therapies. Due to its classical clinical features and its close resemblance to other lesions in the region, it is an entity unlikely to be suspected prior to its histological diagnosis. Despite the benign nature of this tumor, the clinical outcome might be poor. Its treatment may represent a real challenge because it involves critical anatomical areas, leading to high postoperative morbidity and mortality rates. An initial minimally invasive management and adjuvant therapies, such as radiosurgery, in case of symptomatic recurrences, can be effective handling strategies.

A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples.

The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved.

KINFix--A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens.

An optimal fixative should ideally combine the advantages of formalin fixation and freezing, allowing for good preservation of histology and molecular components, easy handling and storage, lack of toxicity, and low costs. Most of these criteria are fulfilled by ethanol-based solutions, and due to our good experience with the commercial RCL2 fixative, reflected by our published single-center trial, we initiated a multicenter ring trial. However, during its course, RCL2 was discontinued on the market. Therefore, we created our own agent, KINFix, composed of the same main constituents as RCL2, and employed it in our laboratory with similar results. Here we present our evaluation of the three fixatives formalin, RCL2, and KINFix from the perspective of histopathology as well as nucleic acid and protein analyses in comparison to fresh frozen tissues together with the multicenter ring trial data for RCL2. We observe that RCL2 and KINFix offer comparable histomorphology and superior template for molecular analyses than formalin. Moreover, KINFix as freely available fixative might overcome some of the difficulties related to the commercial agents. Therefore, we conclude that KINFix might be an attractive complement to formalin in tissue processing and advocate its use in neuropathological practice.

Evidence-Based Diagnostic Algorithm for Glioma: Analysis of the Results of Pathology Panel Review and Molecular Parameters of EORTC 26951 and 26882 Trials.

PURPOSE: With the rapid discovery of prognostic and predictive molecular parameters for glioma, the status of histopathology in the diagnostic process should be scrutinized. Our project aimed to construct a diagnostic algorithm for gliomas based on molecular and histologic parameters with independent prognostic values. METHODS: The pathology slides of 636 patients with gliomas who had been included in EORTC 26951 and 26882 trials were reviewed using virtual microscopy by a panel of six neuropathologists who independently scored 18 histologic features and provided an overall diagnosis. The molecular data for IDH1, 1p/19q loss, EGFR amplification, loss of chromosome 10 and chromosome arm 10q, gain of chromosome 7, and hypermethylation of the promoter of MGMT were available for some of the cases. The slides were divided in discovery (n = 426) and validation sets (n = 210). The diagnostic algorithm resulting from analysis of the discovery set was validated in the latter. RESULTS: In 66% of cases, consensus of overall diagnosis was present. A diagnostic algorithm consisting of two molecular markers and one consensus histologic feature was created by conditional inference tree analysis. The order of prognostic significance was: 1p/19q loss, EGFR amplification, and astrocytic morphology, which resulted in the identification of four diagnostic nodes. Validation of the nodes in the validation set confirmed the prognostic value (P < .001). CONCLUSION: We succeeded in the creation of a timely diagnostic algorithm for anaplastic glioma based on multivariable analysis of consensus histopathology and molecular parameters.

Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients.

Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP+) and promoter methylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) gene. Among these markers, a predictive value was reported in glioblastomas (GBM) for MGMT promoter methylation, in particular in elderly GBM patients. In this study, methylation data from 46 glioma samples with the Illumina 450K platform were obtained and extended using external data to include a total of 247 glioma samples. Methylation analysis of the whole MGMT gene with this platform revealed two strongly survival-associated CpG regions within the promoter and the gene body, which were confirmed in a reported dataset of high grade-gliomas. Methylation at the promoter (CpG 25, cg12981137 and the prognostic model MGMT-STP27) and at the gene body CpG 165 (cg07933035), were significantly associated with better overall survival, and strongly correlated with G-CIMP+ status. In this series, the prognostic value of MGMT methylation at the promoter was not observed in G-CIMP- cases, although around 50 % of them were MGMT-methylated. These results were also obtained in an homogeneously-treated series of chemoradiated G-CIMP- GBMs analyzed by MSP and qMSP, and confirmed in a reported pyrosequencing-analyzed series of gliomas. Interestingly, in contrast to the MGMT promoter, gene body methylation was of prognostic value in G-CIMP-patients older than 65 years. Our study highlights the relevance of the prognostic value of the different regions of methylation throughout the MGMT gene that could be affected by specific G-CIMP profiles and age groups.

Virtual microscopy in the undergraduate teaching of pathology.

BACKGROUND: Little evidence is available concerning the impact of virtual microscopy (VM) in the undergraduate teaching of pathology. We aimed: (1) to determine the impact in student scores when moving from conventional microscopy (CM) to VM; (2) to assess the students' impressions and changes in study habits regarding the impact of this tool. METHODS: We evaluated two groups taking the discipline of pathology in the same course, one using CM and the other VM. The same set of slides used in the CM classes was digitized in a VENTANA iScan HT (Roche Diagnostics, Sant Cugat, Spain) at ×20 and observed by the students using the Virtuoso viewer (Roche Diagnostics). We evaluated the skill level reached by the students with an online test. A voluntary survey was undertaken by the VM group to assess the students' impressions regarding the resource. The day and time of any accession to the viewer were registered. RESULTS: There were no differences between the two groups in their marks in the online test (mean marks for the CM and the VM groups: 9.87 ± 0.34 and 9.86 ± 0.53, respectively; P = 0.880). 86.6% of the students found the software friendly, easy-to-use and effective. 71.6% of the students considered navigation easier with VM than with CM. The most appreciated feature of VM was the possibility to access the images anywhere and at any time (93.3%). 57.5% of the accesses were made on holidays and 41.9% later than 6:00 pm. CONCLUSIONS: Virtual microscopy can effectively replace the traditional methods of learning pathology, providing mobility and convenience to medical students.