Durable tracking anti-SARS-CoV-2 antibodies in cancer patients recovered from COVID-19

Cancer patients are more susceptible to SARS-CoV-2 infection and generally have higher mortality rate. Anti-SARS-CoV-2 IgG is an important consideration for the patients in this COVID-19 pandemic. Recent researches suggested the rapid decay of anti-SARS-CoV-2 antibodies in the general population, but the decline rate of the antibodies in cancer patients was unknown. In this observational study, we reported the clinical features of the 53 cancer patients infected by SARS-CoV-2 from Wuhan, China and tracked the presence of anti-SARS-CoV-2 antibodies in the patients for more than 12 months. We found the duration (days) of anti-SARS-CoV-2 IgG in the patients was significant longer in chemotherapy (mean: 175; range: 75 to 315) and radiotherapy groups (mean: 168; range: 85 to 265) than in non-chemo- or radio-therapy group (mean: 58; range: 21 to 123) after their recovery from COVID-19. We also used single-cell RNA sequencing to track the immunologic changes in a representative patient recovered from COVID-19 and found that CD8 + effective T cells, memory B cells and plasma cells were persistently activated in the patient undergoing chemotherapy. Together, our findings show that chemotherapy and radiotherapy might be beneficial to extend the duration of anti-SARS-CoV-2 IgG.

, and were reported to be more susceptible to SARS-CoV-2 infection and have higher mortality rate compared with regular COVID-19 patients 5,6 . Therefore, cancer patients should be monitored more carefully during the treatment 5 , and the anti-SARS-CoV-2 antibodies are important as they improve the immunity of patients 7,8 .Previous studies suggested the memory B cells (MBCs) against SARS-CoV-2 could be enriched for up for six months in the general convalescent patients 9 , while levels of anti-SARS-CoV-2 IgG antibody rapidly declined as early as three months after infection [10][11][12] . So far, to our knowledge, the duration of the antibodies in cancer patients has not been well established. In this study, we tracked the anti-SARS-CoV-2 antibodies in 53 cancer patients after their recovery from COVID-19 for more than 12 months, aiming to better comprehend the effects of different treatments on the durability of anti-SARS-CoV-2 antibodies and their impact on the immune system of COVID-19 cancer patients.

Results
Anti-SAS-Cov-2 IgG antibody has longer duration in the patients with chemotherapy and radiotherapy. A total of 53 cancer patients (24 women and 19 men) who were infected by SARS-CoV-2 had serial measurements of IgG (Table 1). Infection was confirmed by polymerase chain-reaction assay in all participants. The mean age of patients was 56 years (range: 33 to 78). The mean duration of IgM is 28 days (range: 15 to 65), and the mean duration of IgG is 137 days (range: 21 to 315). There were 17 non-small-cell lung cancer (NSCLC), 6 breast cancer, 5 colon cancer, 5 cervical squamous cell carcinoma (CSCC) and 20 other types of cancer patients in this study (Supplementary Table 1). When we divided the participants into different groups by treatments, we found the duration of IgG was significantly longer in chemotherapy (mean: 175; range: 75 to 315; p < 0.01) and radiotherapy groups (mean: 168; range: 85 to 265; p < 0.01) than in non-chemo-or radiotherapy group (mean: 58; range: 21 to 123) ( Fig. 1) (Table 2). However, we found that the duration of IgG was not significantly correlated with initial IgG levels, gender, cancer type, stage or underlying disease ( Table 2).
The immune system is continuously activated in the chemotherapy patient after the recovery of COVID-19. Interestingly, six participants (11.3%) in our cohorts showed durable presence of the anti-SARS-CoV-2 IgGs, which has already lasted for more 240 days (Table 1). Of them, four received chemotherapies, one received radiotherapy and one received both chemotherapy and radiotherapy after COVID-19 recovery. We collected peripheral blood mononuclear cells (PBMC) from one representative chemotherapy patient recoverd from COVID-19 and performed single-cell RNA sequencing. The uniform manifold approximation and projection (UMAP) (Fig. 2a-c) and trajectory analysis (Fig. 2d)showed the CD8 + effective T cells, memory B cells and plasma cells were persistently activated in this patient after chemotherapy.

Discussion
It has been reported that SARS-CoV-2 could undergo evolution during the treatment of chronic infection [13][14][15][16] . Anti-SARS-CoV-2 IgG antibodies are important for the immunity of the cancer patients 7,8 . In this study, we found that the anti-SARS-CoV-2 IgG antibodies decayed fast in the patients without chemotherapy and radiotherapy, which is consistent with the previous finding in the general population [10][11][12] . However, our findings raise concern that human immunity against SARS-CoV-2 may be long lasting in patients with radiotherapy and  www.nature.com/scientificreports/ chemotherapy. As we know, chemotherapy or radiotherapy can damage the immune system by destroying the hematopoietic stem cells in bone marrow, which may cause immunosuppression 7 . However, cell death caused by the chemotherapy or radiotherapy might also activate the adaptive immune system 17 , resulting in immunogenic cell death effect. Lee et al. 6 found there was no significant effect on mortality for patients with chemotherapy and radiotherapy use within the 4 weeks after testing positive for COVID-19. Hess et al. 18 reported low-dose, wholelung radiation for patients with COVID-19-related pneumonia appeared safe and might be an effective immunomodulatory treatment. Besides, our group 19 and one group in Italy 20 showed that very few patients required treatment interruptions in radiotherapy services, and few patients undergoing radiotherapy were diagnosed with COVID-19 during their treatment course (0.48%, 1 of 209 patients) 19 . Thus, chemotherapy and radiotherapy www.nature.com/scientificreports/ should be safe treatments for the cancer patient recovered from COVID-19. Interestingly, there is also a report that anti-SARS-CoV-2 antibody triggered the anti-tumor immune response in a Hodgkin's lymphoma patient 21 . Therefore, the protective role of IgG antibodies against SARS-CoV-2 in the cancer patient is not only important for them to prevent virus infection, but maybe also beneficial for the cancer treatment. Our study has several limitations. Firstly, this is a multicentric study which performed mainly in two hospitals in Wuhan. We have used different commercial assay Kits to detect anti-SARS-CoV-2 IgG and we could not acquire all the information such as IgG expression levels of patients at each time point. Thus, we mainly focused on the duration but not the expression level of IgG antibody. Secondly, some cancer patients were discharged, died or in unstable physical condition in the process, which resulted in a relatively small sample size. Thirdly, some patients have been used supportive treatments to maintain a normal white blood cell count or hemoglobin level, maintain electrolyte balance and ensure adequate intake. The uncertainties of these different supportive treatments might also affect the duration of anti-SARS-CoV-2 IgG.
In sum, to the best of our knowledge, this study first report that chemotherapy and radiotherapy might provide benefits to prolong the duration of anti-SARS-CoV-2 IgG in human body. This should be important to devise new strategies for cancer treatment and improve antibody therapy in the future. Still, further large-scale investigations on IgG antibodies against SARS-CoV-2 in cancer patients over longer time periods should be done to assess the kinetics of immunity.

Methods
Patient data. We reviewed the medical records, including clinical and treatment data of patients with cancer who were mainly admitted to the Zhongnan Hospital of Wuhan University and Wuhan Tongji Hospital from February 1, 2020, to March 31, 2021. COVID-19 infection was confirmed by polymerase-chain-reaction (PCR) assay. The chat flow of the cancer patients in the study was shown in Supplementary Fig. 1, and the detailed information and clinical features of patients were shown in Supplementary Table 1. During the treatment of patients, venous blood samples were serially collected and analyzed by gold immunochromatography assay (GICA) or enzyme-linked immunosorbent assay (ELISA) or to detect anti-SARS-CoV-2 IgG/IgM. In Tongji hospital, we used one ELISA Kit (EknCov-S1-01, Frdbio bioscience and technology Inc.) and one gold immunochromatography assay (GICA) Kit (200101, Wuhan Easydiagnosis biomedicine Co.Ltd.). In Zhongnan hospital, we mainly used another GICA Kit (20203400240, Zhuhai Livzon Diagnositic Inc.). For the reaction of ELISA 22 , optical density at 450 nm (OD450) was determined with a multifunctional microplate reader. The cutoff for IgG was 0.30 determined by calculating the mean OD450 of a negative serum sample plus 3 SDs. Duration of SARS-CoV-2 antibody among the patients were recorded. All the patients selected in this study were alive before the cutoff date (March 31, 2021), and verbal informed consent was obtained from all the participants. All methods were carried out in accordance with relevant guidelines and regulations.
Single-cell RNA sequencing. Peripheral blood mononuclear cells (PBMCs) were collected from one representative chemotherapy patient using a Ficoll-Hypaque density solution according to the standard density gradient centrifugation methods. This 57-year-old male patient was diagnosed with non-small-cell lung cancer (stage: IIIA) and SARS-CoV-2 infection on February 25, 2020. After recovered from COVID-19, the first blood collection for single-cell RNA sequencing (Singeron) was conducted on April 16, 2020. The patient underwent four chemotherapy cycles (500 mg/m 2 pemetrexed combined with 75 mg/m 2 Nedaplatin) from August to September 2020. The second blood collection for single-cell RNA sequencing was conducted on February 28, 2021. After quality control, we used Seurat v3.8 to do data normalization, dimensional reduction, clustering and calculated differentially express genes (DEGs) among clusters. We identified cell types (15,015 cells) base on DEGs and CellMarker database. Fig. 1 was performed using Prism 7 software (GraphPad La Jolla, USA).

Statistics. Statistical analysis in
Kruskal-Wallis test followed by Mann-Whitney U test were used, p value less than 0.05 was considered to be statistically significant. Linear regression model in Table 2 was performed by the lme4 and lmerTest packages in R version 3.6.1, p value less than 0.05 means statistically significant.
Study approval. This retrospective study was approved by the ethics committee of Wuhan Tongji Hospital (2020370) and Zhongnan Hospital of Wuhan University (2020039).

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary  Information files).