The encounter of tumor and SARS-CoV-2: Pathological ndings in a patient with colon cancer and COVID-19

As coronavirus disease 2019 (COVID-19) spreads worldwide, there have been few reports of infections among the cancer population, although more than six million cases have been conrmed. Here we studied a surgical specimen from a patient with colon cancer and COVID-19 and two tissue microarrays comprising 103 colorectal cancer and 108 enterocyte cases pathologically. The results showed that, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antibody was positively expressed in colon cancer tissues. Moreover, the angiotensin converting enzyme 2 (ACE2) was overexpressed in both colorectal cancer tissue microarray and tumor tissue from this patient, with a positive rate as high as 93.2%. Finally, this case of SARS-CoV-2 infection in colon cancer tumor microenvironment (TME) is a failure of immune homeostasis, due to the decrease in TIA + and Granzyme B+ (GrB) CD8 + T cell proportion and the increase in PD-1 + CD8 + T cell proportion. In summary, we speculate that these three factors may have contributed signicantly to infection risk and severity in the patient.


Introduction
As coronavirus disease 2019 (COVID-19) spreads worldwide, more than six million cases and 378,548 deaths have been reported in 215 countries and regions according to WHO statistics on 2 June 2020.
Previously, statistical evidences supported that the risk of infection and adverse events in the cancer population was higher than that in non-cancer population, and attributed it to systemic immunosuppression in cancer patients. [1] However, some researchers believe that the evidence was not convincing. [2,3] It was also found that ACE2 was highly expressed in the tumor cells by single-cell RNA sequencing. However, the histological evidence for the relationship between ACE2 overexpression in tumor cells, the microenvironment of peritumoral in ammatory cells, and COVID-19 is scarce, [4] In addition, SARS-CoV-2 infection was found in over 50% stool samples, with more than 10% patients exhibiting gastrointestinal (GI) symptoms. Particularly, the SARS-CoV-2 infection rate was 100% in esophagus, stomach, duodenum, and rectum in the endoscopic biopsies of two severe patients with GI symptoms. Therefore, the GI tract may be a potential transmission route and target organ of SARS-CoV-2. [5,6] Recently, immunohistochemical (IHC) staining has con rmed residual SARS-CoV-2 in the pulmonary tissues of a ready-to-discharge patient. [7] Here, we described the histological evidence of tissue in-situ morphology and the TME in a patient with COVID-19 and sigmoid colon cancer.

Case Report
A 62-year-old man was given tracheal intubation for chest tightness after detecting SARS-CoV-2 using real-time (RT-PCR) in sputum samples during a week of hospitalization at a local hospital. The patient was then diagnosed with critical COVID-19 using computed tomography (CT) ( Figure. 2a) after being transferred to the ICU of our hospital on 29 January 2020. On the third day, he showed diarrheal symptoms with bloody stool, which gradually improved in the following ve days. Until the seventh day, the fecal occult blood test reports were negative, but respiratory di culties continued to aggravate. After two weeks, the patient was provided with veno-venous extracorporeal membrane oxygenation (VV-ECMO) treatment and re-developed bloody stool the next day. Abdominal enhancement CT indicated sigmoid colon occupation ( Figure. 2b) and colonoscopy suggested carcinoma. After two tests for SARS-CoV-2 nucleic acid showed negative results and the body temperature returned to normal, the patient underwent radical resection of sigmoid cancer and colostomy on 21 February 2020. Family members living with him, including his wife, son, and daughter-in-law, were released from quarantine after being tested negative for SARS-CoV-2 using nucleic acid tests. The key clinical features are summarized in Fig. 1.
Macroscopic examination showed a 6.5⋅5.0 cm ulcerated and necrotic mass in the middle of the sigmoid colon. Histological examination revealed that the tumor surface and lumen were necrotic, presented a tubular glandular and sylph-like arrangement, and showed the in ltration of peritumor brotic stroma by a large number of in ammatory cells (Fig. 3a).
IHC staining showed that CK20, CDX-2, and mismatch repair marker (MSH2, MSH6, MLH1, and PMS2) expression were positive, while CK7 and CMV expression were negative in the tumor cells. Furthermore, MUC2 was negatively expressed in the tumor cells (Fig. 3b). Compared with that in the surrounding enterocytes, ACE2 expression, located in the cell membrane and cytoplasm of tumor cells, were signi cantly enhanced (Fig. 3c). Two tissue microarray composed of 103 carcinoma tissues and 183 enterocyte were generated to validate that ACE2 expression percentage was very high in both carcinoma and enterocytes (93.2% and 75.4%, respectively), but its intensity was higher in carcinoma than that in controls, the strongly positive ACE2 expression being 73.8% in the tumor, while being only 4.4% in the enterocytes (Table 1). In colon cancer cells, SARS-CoV-2 nucleocapsid expression was localized in the cell membrane and cytoplasm (Fig. 3d), however, all tissue microarray samples showed negative expression.

Discussion
As we know, ACE2, a key receptor for SARS-CoV-2 infection, aids coronavirus attachment to membranes, entry to host cell, and replication; moreover, the attachment of the virus to cell surface ACE2 protects them from immune surveillance mechanisms. [8] Recent studies have revealed that the modi ed S protein of SARS-CoV-2 has a signi cantly higher a nity for ACE2 and is 10-to 20-fold more likely to bind to ACE2 in human cells than that of the SARS-CoV. [9] This study shows that ACE2 were expressed in the cell membrane and cytoplasm of tumoral tissues, but weakly expressed in the enterocytes, which is consistent with ACE2 distribution previously reported. [10] Meanwhile, SARS-CoV-2 nucleocapsid antibody was expressed in the cell membrane and cytoplasm of tumoral tissues. Therefore, initially, we speculated that the strong expression of ACE2 in cancer cells of the patient increases the number of adhesion sites and potential viral load for SARS-CoV-2. This led to increased susceptibility to SARS-CoV-2 and increased severity of manifestations (compared with those who live with him but were not infected, particularly his wife, a 59-year-old woman). This notion is also consistent with the conclusion that transgenic mice were more susceptible to SARS-CoV than were wild-type mice. [11] Furthermore, cell-surface MUC2 is expressed in goblet cells and plays a critical role in the active defense against virus infection. We found that MUC2 was negatively expressed in the carcinoma, which had a defective MUC2 mucin barrier, which might lead to the de ciency in resistance against SARS-CoV-2 infection in the colonic mucosa. [12] Moreover, the intense expression of ACE2 in tumor cells of the patients with colon cancer could further indicate that the GI may be a potential transmission route and target organ of SARS-CoV-2.
Previous reports indicate that CD4 + and CD8 + T cell counts are reduced signi cantly in the patients with COVID-19, suggesting that SARS-CoV-2 suppresses T cell differentiation, which may be related to the severity of COVID-19. [13] Furthermore, the peripheral CD4 + and CD8 T + cell counts are substantially reduced in the blood of the patients with COVID-19, while, mononuclear in ammatory in ltrates, dominated by lymphocytes, are seen in both lungs. [14] Although in ltrated CD4 + and CD8 + T cells were observed in the carcinoma tissue, colonic carcinoma environment is a particular circumstance where the immune response could be ine cient or unresponsive, resulting in uncontrolled growth of the carcinoma cells. The activated TIA-1 + and GrB + CD8 + T cells were limited in number in colonic carcinoma, while the PD-1 + CD8 + T cells increased, which is not only one of the signs of T cell failure, but also a common pathway inhibitor for CD8 T cells following respiratory virus infection. [15] Therefore, a failure of immune homeostasis occurs, which consequently causes poor reactivity in the TILs. Substantial and interactive impacts between carcinoma and immune cells create a microenvironment suitable for SARS-CoV-2 infection.
On the contrary, COVID-19 causes multiple organ dysfunction syndrome rather than respiratory failure, which might be attributable to the widespread distribution of ACE2 in multiple organs. ACE2 overexpression in colonic carcinoma might be the reason for the obvious symptom in the GI.
In summary, pathologically, we observed, for the rst time, a positive expression of SARS-CoV-2 nucleocapsid antibody in colon cancer tissues. Moreover, ACE2, a key receptor for SARS-CoV-2, was overexpressed in the colon tumor cells of the patient with COVID-19 and the TME may be more suitable for SARS-CoV-2 survival. we speculate that these factors may have contributed signi cantly to infection risk and severity in the patient. We also provided in situ morphological evidence indicating that patients with cancer may have increased SARS-CoV-2 susceptibility and severe infection and the GI may be a potential transmission route and target organ for SARS-CoV-2.

Declarations
Con ict of interests The authors have disclosed that they have no signi cant relationships with or nancial interest in any commercial companies pertaining to this article.  The clinical course of the patient with colon cancer and COVID-19. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. VV-ECMO=veno-venous extracorporeal membrane oxygenation.

Figure 2
Transverse thin-section computed tomography (CT) scans in the patient with colon cancer and COVID-19. A. Chest CT shows diffuse ground-glass opacities in both lungs, giving a white lung appearance, with air bronchograms. B. Abdominal enhancement CT reveals that the wall of the middle sigmoid colon thickened markedly to form an ulcerated mass with marked enhancement and rough serous surface.

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