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Abstract
Background Persistence of SARS-CoV-2 virus particles in recovered COVID-19 patients remains a challenge as we continue to fight the ongoing pandemic. For instance, despite three negative consecutive nasopharyngeal swab PCR tests, residual SARS-CoV-2 was reported in the lungs of a deceased patient.1 Moreover, viral RNA could also be detected in rectal tissues that were obtained during incubation period.2 To date, there is no data regarding residual viral particles present in tissues from recovered COVID-19 patients. Hereby, we reported our findings of SARS-CoV-2 viral antigen in liver tissues from a recovered COVID-19 patient. These findings raise concern for potential transmissibility in recovered individuals.
Methods A 49-year-old South Asian male diagnosed with COVID-19 in June 2020, with incidental discovery of hepatitis B virus (HBV)-associated R0 Grade 2 hepatocellular carcinoma (HCC), was consented for our study. He did not develop significant acute respiratory symptoms throughout the course of the disease. He underwent curative resection of HCC 85 days after being tested COVID-19 negative where his blood, normal tissue and tumour samples were obtained for further analysis (figure 1). We performed deep immunopathological profiling on the specimens using multiplex immunohistochemistry and 25-colour flow cytometry to study SARS-CoV-2-elicited immune response.
Results Multiplex immunohistochemistry detected SARS-CoV-2 nucleocapsid protein only in adjacent normal liver tissue but not within tumour core (figure 2). We also observed SARS-CoV-2 in some immune cells such as sinusoidal Kupffer cells (figure 2). Additionally, upon stimulation with SARS-CoV-2 peptides, we successfully elicited SARS-CoV-2-specific memory response which is distinct from the response upon challenge with HBV peptides. These findings were similar to our previous discovery in a patient with colorectal adenocarcinoma where we have shown viral antigen detection, validated with PCR to detect viral RNA, as well as the detection of SARS-CoV-2 memory-like T cells in situ (figure 2). Deep profiling of the samples is on-going with single-cell analysis and digital spatial profiling.
Study design, methodology and brief summary of the findingsBlood, normal tissue and tumour samples were obtained from a 49-year-old South Asian male who was diagnosed with COVID-19 and hepatocellular carcinoma. Normal tissue and tumour samples were analysed with multiplex immunohistochemistry, while dissociated cells from blood and tissue samples were subjected to SARS-CoV-2 peptide stimulation and analysed with 25-colour flow cytometry. Multiplex immunohistochemistry detected SARS-CoV-2 proteins in both tumour and adjacent normal tissues, while flow cytometry identified distinct immune microenvironment involving memory-like T cells.
Immunohistochemical staining of the SARS-CoV-2 nucleocapsid protein and immune profiling with 25-colour flow cytometry in normal colon and liver tissue a, Liver tissues were immunostained with SARS-CoV-2 nucleocapsid protein (NP), nuclei were counterstained with haematoxylin. Positive SARS-CoV-2 nucleocapsid staining in benign hepatocytes and sinusoidal Kupffer cells. Scale bar represents 50μm. b, Multiplex immunohistochemistry of normal liver tissue. From left to right, top to bottom: SARS-CoV-2 nucleocapsid (green), SARS-CoV-2 nucleocapsid (green) with CD14 (red), SARS-CoV-2 nucleocapsid (green) with CD68 (pink) and composite. Co-localisation were observed as shown by the white arrows. Scale bar represents 100μm. c, Colon tissues were immunostained with SARS-CoV-2 nucleocapsid protein, nuclei were counterstained with haematoxylin. Positive SARS-CoV-2 nucleocapsid staining in colonic crypts, with granular supranuclear cytoplasmic pattern. Scale bar represents 50μm. d, Multiplex immunohistochemistry of colon tissue. From left to right, top to bottom: DAPI (blue), CD3 (magenta), CD38 (green), granzyme B (yellow), interferon-gamma (red) and composite. Co-localisation was observed as shown by the white arrows. Scale bar represents 100μm, Magnification x200. e, Flow cytometry immune profiling of blood from colorectal cancer patient with COVID-19 following stimulation with SARS-CoV-2 peptides. Highlighted populations showed CD3 cells expressing CD38, supporting the CD3+ CD38+ co-localization findings observed in (c).
Conclusions We believe this is the first immune profiling report of the in situ tumour microenvironment in a cancer patient with COVID-19. Our findings demonstrated the presence of viral proteins in the liver despite negative swab test result and the ability to elicit ex vivo SARS-CoV-2-specific immune responses through peptide stimulation assays. We also detected same immune cell phenotypes in situ in the cancer tissues. Taken together, we propose caution when handling tissues from patients who have a recent history of COVID-19, particularly during aerosol-generating procedures such as ultrasonic dissection surgery.
Ethics Approval This study was approved by Centralised Institutional Review Board of SingHealth, approval number 2019/2653.
Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.
References
Yao XH, He ZC, Li TY, Zhang HR, Wang Y, Mou H, et al. Pathological evidence for residual SARS-CoV-2 in pulmonary tissues of a ready-for-discharge patient. Cell Res 2020;30(6):541-3.
Qian Q, Fan L, Liu W, Li J, Yue J, Wang M, et al. Direct evidence of active SARS-CoV-2 replication in the intestine. Clin Infect Dis 2020.
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