Neuropathology of COVID-19 (neuro-COVID): clinicopathological update.

Coronavirus disease 2019 (COVID-19) is emerging as the greatest public health crisis in the early 21stcentury. Its causative agent, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), is an enveloped single stranded positive-sense ribonucleic acid virus that enters cells via the angiotensin converting enzyme 2 receptor or several other receptors. While COVID-19 primarily affects the respiratory system, other organs including the brain can be involved. In Western clinical studies, relatively mild neurological dysfunction such as anosmia and dysgeusia is frequent (~70-84%) while severe neurologic disorders such as stroke (~1-6%) and meningoencephalitis are less common. It is unclear how much SARS-CoV-2 infection contributes to the incidence of stroke given co-morbidities in the affected patient population. Rarely, clinically-defined cases of acute disseminated encephalomyelitis, Guillain-Barré syndrome and acute necrotizing encephalopathy have been reported in COVID-19 patients. Common neuropathological findings in the 184 patients reviewed include microglial activation (42.9%) with microglial nodules in a subset (33.3%), lymphoid inflammation (37.5%), acute hypoxic-ischemic changes (29.9%), astrogliosis (27.7%), acute/subacute brain infarcts (21.2%), spontaneous hemorrhage (15.8%), and microthrombi (15.2%). In our institutional cases, we also note occasional anterior pituitary infarcts. COVID-19 coagulopathy, sepsis, and acute respiratory distress likely contribute to a number of these findings. When present, central nervous system lymphoid inflammation is often minimal to mild, is detected best by immunohistochemistry and, in one study, indistinguishable from control sepsis cases. Some cases evince microglial nodules or neuronophagy, strongly supporting viral meningoencephalitis, with a proclivity for involvement of the medulla oblongata. The virus is detectable by reverse transcriptase polymerase chain reaction, immunohistochemistry, or electron microscopy in human cerebrum, cerebellum, cranial nerves, olfactory bulb, as well as in the olfactory epithelium; neurons and endothelium can also be infected. Review of the extant cases has limitations including selection bias and limited clinical information in some cases. Much remains to be learned about the effects of direct viral infection of brain cells and whether SARS-CoV-2 persists long-term contributing to chronic symptomatology.

Effect of repetitive transcranial magnetic stimulation (rTMS) treatment of major depressive disorder (MDD) on cognitive control.

BACKGROUND: Major Depressive Disorder (MDD) is commonly accompanied by cognitive control dysfunction that may persist after remission of clinical symptoms with antidepressant medication treatment. Repetitive Transcranial Magnetic Stimulation (rTMS) is an effective treatment alternative for medication-resistant MDD. In this study, we investigated whether rTMS treatment had a beneficial effect not only on depressive symptoms, but on also cognitive control dysfunction. METHODS: 77 subjects with MDD received a 30-session treatment course of 10 Hz rTMS administered at the left dorsolateral prefrontal cortex (DLPFC). Treatment efficacy was assessed using the Inventory of Depressive Symptomatology Self-Rated (IDS-SR) before and after treatment, with clinical response defined as 50% or greater decrease in the IDS-SR score at treatment 30. Cognitive control function was assessed before and after treatment using the Stroop word-color interference task. We examined changes in Stroop accuracy and reaction time for congruent and incongruent trials, as well as in relation to changes in depressive symptoms. RESULTS: Performance accuracy improved particularly for the rTMS responders in the incongruent condition, with older subjects benefitting most from the rTMS treatment. Improvement in reaction times was positively associated with clinical improvement, especially in the incongruent condition. LIMITATIONS: We used a single cognitive task in a naturalistic setting without control for individual rTMS treatment parameters or concomitant medication. CONCLUSIONS: Overall, these results indicate that rTMS treatment for MDD has beneficial effects on psychomotor speed and cognitive control. Future studies should extend these findings to larger patient populations and other cognitive domains.

Use of Phenobarbital in Delirium Tremens.

The standard of care for alcohol withdrawal centers on the use of escalating doses of benzodiazepines until clinical improvement is achieved. However, there is no established standard in the care of patients with severe alcohol withdrawal and delirium tremens that is refractory to benzodiazepine therapy. One potential therapy that is gaining traction is the use of phenobarbital, which may be mechanistically superior to benzodiazepines in treating delirium tremens because of its effects on GABA and N-methyl-D-aspartate receptors. The dosing of phenobarbital and its subsequent taper, however, is still unclear and the side effect profile is not well characterized. In this case report, we present the case of a 37-year-old Hispanic male who presented with alcohol withdrawal and subsequent delirium tremens who was treated with phenobarbital with positive clinical response and minimal side effects.

Tumor-suppressive miR148a is silenced by CpG island hypermethylation in IDH1-mutant gliomas.

PURPOSE: IDH1/2-mutant gliomas harbor a distinct glioma-CpG island methylation phenotype (G-CIMP) that may promote the initiation and progression of secondary pathway gliomas by silencing tumor-suppressive genes. The potential role of tumor-suppressive microRNAs (miRNA; miR) in this process is not understood. EXPERIMENTAL DESIGN: To identify potential tumor-suppressive miRNA hypermethylated in glioma, the methylation profiles of IDH1/2(WT) gliomas (n = 11) and IDH1(MUT) glioma (n = 20) were compared by using massively parallel reduced representation bisulfite sequencing (RRBS). The methylation status of selected miRNA was validated by using targeted bisulfite sequencing (BiSEQ) in a large cohort of glioma tissue samples including 219 IDH1(WT) and 72 IDH1/2(MUT) samples. The expression of selected miRNAs was determined by using the TaqMan qPCR. Functional analyses of miR148a were conducted and target genes were identified. RESULTS: We identify miR148a as a novel, G-CIMP-associated miRNA whose methylation is tightly correlated with IDH1 mutation and associated with improved survival in patients with malignant glioma. We confirm that downregulation of miR148a can occur via DNA methylation. We demonstrate that IDH1 mutation provides a mechanism of miR148a methylation and downregulation, and that restoration of miR148a reduced tumorigenic properties of glioma cells, possibly by targeting DNMT1. CONCLUSIONS: We identify miR148a as a novel G-CIMP-associated miRNA, and provide results suggesting that miR148a restoration may have therapeutic implications.

The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings.

Well preserved frozen biospecimens are ideal for evaluating the genome, transcriptome, and proteome. While papers reviewing individual aspects of frozen biospecimens are available, we present a current overview of experimental data regarding procurement, storage, and quality assurance that can inform the handling of frozen biospecimens. Frozen biospecimen degradation can be influenced by factors independent of the collection methodology including tissue type, premortem agonal changes, and warm ischemia time during surgery. Rapid stabilization of tissues by snap freezing immediately can mitigate artifactually altered gene expression and, less appreciated, protein phosphorylation profiles. Collection protocols may be adjusted for specific tissue types as cellular ischemia tolerance varies widely. If data is not available for a particular tissue type, a practical goal is snap freezing within 20min. Tolerance for freeze-thaw events is also tissue type dependent. Tissue storage at -80°C can preserve DNA and protein for years but RNA can show degradation at 5years. For -80°C freezers, aliquots frozen in RNAlater or similar RNA stabilizing solutions are a consideration. It remains unresolved as to whether storage at -150°C provides significant advantages relative to that at -80°C. Histologic quality assurance of tissue biospecimens is typically performed at the time of surgery but should also be conducted on the aliquot to be distributed because of tissue heterogeneity. Biobanking protocols for blood and its components are highly dependent on intended use and multiple collection tube types may be needed. Additional quality assurance testing should be dictated by the anticipated downstream applications.

A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories.

UNLABELLED: Frozen biospecimens are crucial for translational research and contain well-preserved nucleic acids and protein. However, the risks of freezer failure as well as space, cost, and environmental concerns of frozen biospecimens are substantial. OBJECTIVE: The purpose of the study was to review the current status of room temperature biospecimen storage. METHODS: We searched Pubmed and vendor websites to identify relevant information. RESULTS: Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can now robustly preserve fresh tissue for up to 7days at room temperature. For longer term storage, commercial vendors of chemical matrices claim real time stability of nucleic acids of over 2 years and their accelerated aging studies to date suggest stability for 12years for RNA and 60years for DNA. However, anatomic pathology biorepositories store mostly frozen tissue rather than nucleic acids. Small quantities of tissue can be directly placed on some chemical matrices to stabilize DNA, however RNA and proteins are not preserved. Current lyophilization approaches can preserve histomorphology, DNA, RNA, and proteins though RNA shows moderate degradation after 1-2years. Formalin-free fixatives show improved but varying abilities to preserve nucleic acids and face validation as well as cost barriers in replacing FFPE specimens. The paraffin embedding process can degrade RNA. CONCLUSION: Development of robust long-term room temperature biospecimen tissue storage technology can potentially reduce costs for the biomedical community in the face of growing targeted therapy needs and decreasing budgets.

Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage.

Frozen tissue, a gold standard biospecimen, can yield well preserved nucleic acids and proteins after over a decade but is vulnerable to thawing and has substantial fiscal, spatial, and environmental costs. A long-term room temperature biospecimen storage alternative that preserves broad analytical utility can potentially empower tissue-based research. As there is scant data on the analytical utility of lyophilized brain tumor biospecimens, we evaluated lyophilized (freeze-dried) samples stored for 1 year at room temperature. Lyophilized tumor tissue processed into paraffin sections produced good histology. Yields of extracted DNA, RNA, and protein approximated those of frozen tissue. After 1 year, lyophilized samples yielded high molecular weight DNA that permitted copy number variation analysis, IDH 1 mutation detection, and MGMT promoter methylation PCR. A 27 % decrease in RIN scores over the 1 year suggests that RNA degradation was inhibited though incompletely. Nevertheless, RT-PCR studies on lyophilized tissue performed similarly to frozen tissue. In contrast to FFPE tissues where protein bands were absent or shifted to a lower molecular weight, lyophilized samples showed similar protein bands as frozen tissue on SDS-PAGE analysis. Lyophilized tissue performed similarly to frozen tissue for Western blots and enzyme activity assays. Immunohistochemistry of lyophilized tissue that were processed into FFPE blocks often required longer incubation times for staining than standard FFPE samples but generally provided robust antigen detection. This preliminary study suggests that lyophilization has promise for long-term room temperature storage while permitting varied tests; however, further work is required to better stabilize nucleic acids particularly RNA.

Combined analysis of O6-methylguanine-DNA methyltransferase protein expression and promoter methylation provides optimized prognostication of glioblastoma outcome.

BACKGROUND: Promoter methylation of the DNA repair gene, O-6-methylguanine-DNA methyltransferase (MGMT), is associated with improved treatment outcome for newly diagnosed glioblastoma (GBM) treated with standard chemoradiation. To determine the prognostic significance of MGMT protein expression as assessed by immunohistochemistry (IHC) and its relationship with methylation, we analyzed MGMT expression and promoter methylation with survival in a retrospective patient cohort. METHODS: We identified 418 patients with newly diagnosed GBM at University of California Los Angeles Kaiser Permanente Los Angeles, nearly all of whom received chemoradiation, and determined MGMT expression by IHC, and MGMT promoter methylation by methylation-specific PCR (MSP) and bisulfite sequencing (BiSEQ) of 24 neighboring CpG sites. RESULTS: With use of the median percentage of cells staining by IHC as the threshold, patients with <30% staining had progression-free survival (PFS) of 10.9 months and overall survival (OS) of 20.5 months, compared with PFS of 7.8 months (P < .0001) and OS of 16.7 months (P < .0001) among patients with ≥30% staining. Inter- and intrareader correlation of IHC staining was high. Promoter methylation status by MSP was correlated with IHC staining. However, low IHC staining was frequently observed in the absence of promoter methylation. Increased methylation density determined by BiSEQ correlated with both decreased IHC staining and increased survival, providing a practical semiquantitative alternative to MSP. On the basis of multivariate analysis validated by bootstrap analysis, patients with tandem promoter methylation and low expression demonstrated improved OS and PFS, compared with the other combinations. CONCLUSIONS: Optimal assessment of MGMT status as a prognostic biomarker for patients with newly diagnosed GBM treated with chemoradiation requires determination of both promoter methylation and IHC protein expression.

Overexpression of isocitrate dehydrogenase mutant proteins renders glioma cells more sensitive to radiation.

Mutations in isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) are found in a subset of gliomas. Among the many phenotypic differences between mutant and wild-type IDH1/2 gliomas, the most salient is that IDH1/2 mutant glioma patients demonstrate markedly improved survival compared with IDH1/2 wild-type glioma patients. To address the mechanism underlying the superior clinical outcome of IDH1/2 mutant glioma patients, we investigated whether overexpression of the IDH1(R132H) protein could affect response to therapy in the context of an isogenic glioma cell background. Stable clonal U87MG and U373MG cell lines overexpressing IDH1(WT) and IDH1(R132H) were generated, as well as U87MG cell lines overexpressing IDH2(WT) and IDH2(R172K). In vitro experiments were conducted to characterize baseline growth and migration and response to radiation and temozolomide. In addition, reactive oxygen species (ROS) levels were measured under various conditions. U87MG-IDH1(R132H) cells, U373MG-IDH1(R132H) cells, and U87MG-IDH2(R172K) cells demonstrated increased sensitivity to radiation but not to temozolomide. Radiosensitization of U87MG-IDH1(R132H) cells was accompanied by increased apoptosis and accentuated ROS generation, and this effect was abrogated by the presence of the ROS scavenger N-acetyl-cysteine. Interestingly, U87MG-IDH1(R132H) cells also displayed decreased growth at higher cell density and in soft agar, as well as decreased migration. Overexpression of IDH1(R132H) and IDH2(R172K) mutant protein in glioblastoma cells resulted in increased radiation sensitivity and altered ROS metabolism and suppression of growth and migration in vitro. These findings provide insight into possible mechanisms contributing to the improved outcomes observed in patients with IDH1/2 mutant gliomas.

A review of radio frequency identification technology for the anatomic pathology or biorepository laboratory: Much promise, some progress, and more work needed.

Patient safety initiatives throughout the anatomic laboratory and in biorepository laboratories have mandated increasing emphasis on the need for accurately identifying and tracking biospecimen assets throughout their production lifecycle and for archiving/retrieval purposes. However, increasing production volume along with complex workflow characteristics, reliance on manual production processes, and required asset movement to disparate destinations throughout asset lifecycles continue to challenge laboratory efforts. Radio Frequency Identification (RFID) technology, use of radio waves to communicate data between electronic tags attached to objects and a reader, shows significant potential to facilitate and overcome these hurdles. Advantages over traditional barcode labeling include readability without direct line-of-sight alignment to the reader, ability to read multiple tags simultaneously, higher data storage capacity, faster data transmission rate, and capacity to perform multiple read-writes of data to the tag. Most importantly, use of radio waves decreases the need to manually scan each asset, and at each step, identification or tracking event is needed. Temperature monitoring by on-board sensors and three-dimensional position tracking are additional potential benefits of using RFID technology. To date, barriers to implementation of RFID systems in the anatomic laboratory include increased associated costs of tags and readers, system software, data security concerns, lack of specific data standards for stored information, and potential for technological obsolescence during decades of specimen storage. Novel RFID production techniques and increased production capacity are projected to lower costs of some tags to a few cents each. Potentially, information security concerns can be addressed by techniques such as shielding, data encryption, and tag pseudonyms. Commitment by stakeholder groups to develop RFID tag data standards for anatomic pathology and biorepository laboratories could avoid or mitigate the "islands of data" dilemma presented by barcode usage where there are innumerable standards and a consequent paucity of hardware or software "plug and play" interoperability. Work remains to be done to establish the durability and appropriate shielding of individual tag types for use in harsh laboratory environmental conditions, and for long-term archival storage. Finally, given the requirements for long-term storage of biospecimen assets, consideration should be given to ways of mitigating data isolation due to eventual technological obsolescence of a particular RFID technology or software.

Tolerance testing of passive radio frequency identification tags for solvent, temperature, and pressure conditions encountered in an anatomic pathology or biorepository setting.

BACKGROUND: Radio frequency identification (RFID) tags have potential for use in identifying and tracking biospecimens in anatomic pathology and biorepository laboratories. However, there is little to no data on the tolerance of tags to solutions, solvents, temperatures, and pressures likely to be encountered in the laboratory. The functioning of the Hitachi Mu-chip RFID tag, a candidate for pathology use, was evaluated under such conditions. METHODS: The RFID tags were affixed to cryovials containing tissue or media, glass slides, and tissue cassettes. The tags were interrogated for readability before and after each testing condition or cycle. Individual tags were subjected to only one testing condition but for multiple cycles. Testing conditions were: 1) Ten wet autoclave cycles (121°C, 15 psi); 2) Ten dry autoclave cycles (121°C, 26 psi); 3) Ten tissue processor cycles; 4) Ten hematoxylin and eosin (H&E) staining cycles; 5) Ten antigen retrieval pressure cooker cycles (125°C, 15 psi); 6) 75°C for seven days; 7) 75-59 °C day/night cycles for 7 days; 8) -80°C, -150°C, or -196°C for 12 months; 9) Fifty freeze-thaw cycles (-196°C to 22°C). RESULTS: One hundred percent of tags exposed to cold temperatures from -80 to -196 °C (80 tags, 1120 successful reads), high temperatures from 52 to 75°C (40 tags, 420 reads), H & E staining (20 tags, 200 reads), pressure cooker antigen retrieval (20 tags, 200 reads), and wet autoclaving (20 tags, 200 reads) functioned well throughout and after testing. Of note, all 20 tested tags tolerated 50 freeze-thaw cycles and all 60 tags subjected to sustained freezing temperatures were readable after 1 year. One dry autoclaved tag survived nine cycles but failed after the tenth. The remaining 19 tags were readable after all 10 dry autoclave cycles. One tag failed after the first tissue processing cycle while the remaining 19 tags survived all 10 tissue processing cycles. CONCLUSIONS: In this preliminary study, these RFID tags show a high-degree of tolerance to tested solutions, solvents, temperature, and pressure conditions. However, a measurable failure rate is detectable under some circumstances and redundant identification systems such as barcodes may be required with the deployment of RFID systems. We have delineated testing protocols that may be used as a framework for preliminary assessments of candidate RFID tag tolerance to laboratory conditions.