A case of persistent severe sequelae of COVID-19 infection: potential role in sudden death?

Depending on the stage of the disease, autopsy findings of COVID-19 may include a spectrum of cardiopulmonary pathologies including alveolar hyaline membrane formation, vascular thrombosis, and intracardiac thrombi. Identification of a COVID-19 positive decedent in the absence of clinical history relies primarily on post-mortem nasopharyngeal (NP) or oropharyngeal (OP) swabs for real time polymerase chain reaction (RT-PCR). In the absence of definitive microbiology testing, post-mortem computed tomography (PMCT) may be a powerful adjunct tool for screening. Persistence of pathological changes may prolong physiological alterations and increase the risk of cardiopulmonary compromise. This current case outlines the forensic presentation, utilization of screening tools including PMCT, and the autopsy findings of a recent toxicology related sudden death case in the context of severe sequelae of COVID-19 pneumonia. This case demonstrates the limitation of NP and OP swabs in the post-mortem setting, the value of PMCT as an adjunct screening tool, and raises the consideration of COVID-19 sequelae as a potential contributing risk factors in sudden death cases in the community.

Development of a multiplex immuno-oncology biomarker and digital pathology workflow for assessment of urothelial carcinoma.

BACKGROUND: Immune checkpoint inhibitors (ICI) therapies have demonstrated significant benefit in the treatment of many tumors including high grade urothelial cancer (HGUC) of the bladder. However, variability in patients' clinical responses highlights the need for biomarkers to aid patient stratification. ICI relies on an intact host immune response. In this context, we hypothesize that key players in the antitumor immune response such as markers of activated cytotoxic T lymphocytes (CD8, granzyme-B) and immune suppression (FOXP3) may help to identify patients who will derive the greatest therapeutic benefit from ICI. A major obstacle for deployment of such a strategy is the limited quantities of tumor-derived biopsy material. Therefore, in this technical study, we develop a multiplex biomarker with digital workflow. We explored the (1) concordance of conventional single stain results using digital image analysis, and (2) agreement between digital scoring versus manual analysis. METHODS: (1) For concordance study of single and multiplex stains, triplicate core tissue microarrays of 207 muscle invasive, HGUC of bladder had sequential 4-micron sections cut and stained with CD8, FOXP3 and granzyme-B. An inhouse developed tri-chromogen multiplex immunohistochemistry (m-IHC) assay consisting of CD8 (green), granzyme B (brown), and FOXP3 (red) was used to stain the next sequential tissue section. (2) Agreement between manual and digital analysis was performed on 19 whole slide sections of HGUC cystectomy specimens. All slides were scanned using Aperio ScanScope AT Digital Scanner at 40X. Quantitative digital image analysis was performed using QuPath version 0.2.3 open-source software. Scores from triplicate cores were averaged for each HGUC specimen for each marker. Intraclass correlation coefficients were used to compare percent positive cells between the single- and multi-plex assays. Lin's concordance correlation coefficients were used for manual versus digital analysis. RESULTS AND CONCLUSIONS: m-IHC offers significant advantages in characterizing the host immune microenvironment particularly in limited biopsy tissue material. Utilizing a digital image workflow resulted in significant concordance between m-IHC and individual single stains (p < 0.001 for all assessments). Moderate to good agreements were achieved between manual and digital scoring. Our technical work demonstrated potential uses of multiplex marker in assessing the host immune status and could be used in conjunction with PD-L1 as a predictor of response to ICI therapy.