475?Incidental finding of colorectal cancer in a COVID-19 patient, followed by deep profiling of SARS-CoV-2-associated immune landscape and tumour microenvironment

摘要

Background Reports suggest that cancer patients may be more vulnerable to COVID-19, with increased disease severity and higher mortality rate. 1–3 Although this is likely multifactorial, the exact pathogenesis has not been clearly elucidated. Studies have shown increased ACE2 expression in tumours as compared to normal tissues, 4 5 thereby providing increased viral binding. Moreover, other mechanisms of cancer immunotherapy including treatment- and disease-related immunosuppression and functional exhaustion have been reported in patients with concomitant cancer and COVID-19; contributing to greater COVID-19 disease severity. 6–8 There is still much to be revealed about the interplay between COVID-19, cancer and the immune system. These insights will give us greater understanding of the immunopathological processes underlying COVID-19 in cancer patients and their clinical relevance. Methods A 45-year-old South Asian male diagnosed with COVID-19, with incidental discovery of stage II T3N0 caecal adenocarcinoma was consented for our study. The patient had experienced mild symptoms throughout the course of the disease, and underwent laparoscopic right hemicolectomy 10 days after recovery from COVID-19. His blood, lymph nodes, normal tissue and tumour samples were obtained for further analysis (figure 1). Multiplex immunohistochemistry was performed to understand SARS-CoV-2-associated tumour immune microenvironment. Moreover, to simulate ex vivo SARS-CoV-2 infection, dissociated cells from blood, lymph nodes, and tissue samples were stimulated with SARS-CoV-2 peptides or control for 16 hours. This was followed by 25-colour flow cytometry analysis for immune markers and cytokines. We then compared unstimulated with stimulated cells to study SARS-CoV-2-elicited immune response. Results Multiplex immunohistochemistry demonstrated upregulated expression of ACE2 in the tumour as compared to adjacent normal tissue, whilst SARS-CoV-2 was detected only in adjacent normal tissue but not within the tumour (figure 2). We also observed SARS-CoV-2 in other organs such as appendix and lymph nodes; and the presence of tertiary lymphoid structure, abundant T cells and NK cells within the proximity of the tumour (figure 2). Additionally, upon stimulation with SARS-CoV-2 peptides, we successfully elicited SARS-CoV-2-specific CD4 T cells expressing immune markers such as granzyme B, TNF-α and IFN-γ (figure 3). Deep profiling of the samples is on-going with single-cell sequencing and digital spatial profiling. Abstract 475 Figure 1 Study design, methodology and brief summary of the findingsBlood, lymph nodes, normal tissue and tumour samples were obtained from a 45-year-old South Asian male who was diagnosed with COVID-19 and caecal adenocarcinoma. Lymph nodes, normal tissue and tumour samples were analysed with multiplex immunohistochemistry, while dissociated cells from blood, lymph nodes 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 only in adjacent normal tissue but not within the tumour. Exhausted tumour-infiltrating T cells were also detected. Flow cytometry revealed CD4 T cells expressing IFN-γ and granzyme B Abstract 475 Figure 2 Multiplex immunohistochemistry of tissue samples(A) Multiplex immunohistochemistry of normal colon tissue. From left to right: SARS-CoV-2 nucleocapsid (green), CD3 (red), CD56 (cyan) and FOXP3 (white), representative of SARS-CoV-2 virus, T cells, NK cells and regulatory T cells respectively. (B) Multiplex immunohistochemistry of tertiary lymphoid structure. First row from left to right: PD-L1 (green), CD3 (orange), CD68 (red) and DAPI (blue). Second row from left to right: CD8 (magenta), cytokeratin (white), FOXP3 (cyan) and composite Abstract 475 Figure 3 Cytokine profiling with 25-colour flow cytometry panelBlood cells were incubated with SARS-CoV-2 peptides or control for 16 hours. This was followed by 25-colour flow cytometry panel with immune markers and cytokines. Both gated populations were observed to be increased after stimulation with SARS-CoV-2 peptides, suggesting that they might be SARS-CoV-2-specific T cells. Further gating on the populations showed that they were CD4 T cells expressing granzyme B, with high (population 2) or moderate (population 1) TNF-α and IFN-γ expressions Conclusions We believe this is the first report of immune profiling of in situ tumour microenvironment in a cancer patient with COVID-19. Our findings showed the presence of viral proteins in several tissues despite negative swab test result, and the ability to elicit ex vivo SARS-CoV-2-specific T cell responses through peptide stimulation experiments. 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 accompanyin