SARS-CoV-2 Vaccine Immunogenicity in Patients with Gastrointestinal Cancer Receiving Systemic Anti-Cancer Therapy

Abstract Introduction Patients with gastrointestinal (GI) cancers have an increased risk of serious complications and death from SARS-CoV-2 infection. The immunogenicity of vaccines in patients with GI cancers receiving anti-cancer therapies is unclear. We conducted a prospective study to evaluate the prevalence of neutralizing antibodies in a cohort of GI cancer patients receiving chemotherapy following SARS-CoV-2 vaccination. Materials and Methods Between September 2020 and April 2021, patients with cancer undergoing chemotherapy were enrolled. At baseline (day 0), days 28, 56, and 84, we assessed serum antibodies to SARS-CoV-2 spike (anti-S) and anti-nucleocapsid (anti-NP) and concomitantly assessed virus neutralization using a pseudovirus neutralization assay. Patients received either the Pfizer/BioNTech BNT162b2, or the Oxford/AstraZeneca ChAdOx1 vaccine. Results All 152 patients enrolled had a prior diagnosis of cancer; colorectal (n = 80, 52.6%), oesophagogastric (n = 38, 25.0%), and hepato pancreatic biliary (n = 22, 12.5%). Nearly all were receiving systemic anti-cancer therapy (99.3%). Of the 51 patients who did not receive a vaccination prior to, or during the study, 5 patients had detectable anti-NP antibodies. Ninety-nine patients received at least one dose of vaccine prior to, or during the study. Within 19 days following the first dose of vaccine, 30.0% had anti-S detected in serum which increased to 70.2% at days 20-39. In the 19 days following a second dose, anti-S positivity was 84.2% (32/38). However, pseudovirus neutralization titers (pVNT80) decreased from days 20 to 39. Conclusion Despite the immunosuppressive effects of chemotherapy, 2 doses of SARS-CoV-2 vaccines are able to elicit a protective immune response in patients’ ongoing treatment for gastrointestinal cancers. Decreases in pseudoviral neutralization were observed after 20-39 days, re-affirming the current recommendation for vaccine booster doses. Clinical Trial Registration Number NCT04427280.


Introduction
Globally, COVID-19 caused by the SARS-CoV-2 virus has led to over 440 million infections and approximately 6 million deaths to date. 1 There is substantial evidence patients with cancer are at a high risk of severe complications and poor outcomes from SARS-CoV-2 infection. 2,3 It is unclear if an immunity to COVID-19 is maintained during chemotherapy and if patients undergoing cytotoxic chemotherapy are able to mount protective immune responses to SARS-CoV-2. These factors have important consequences for the health of patients and the control of SARS-CoV-2 transmission within healthcare facilities emphasizing, the need to establish the effectiveness of SARS-CoV-2 vaccines in patients with cancer.
The Pfizer/BioNTech BNT162b2 and Oxford-AstraZeneca ChAdOx1 nCOV-19 vaccines induce immune responses to the SARS-CoV-2 spike protein and are highly effective in preventing severe complication and death from COVID-19. 4,5 As patients with malignancy were excluded from these vaccine trials, there is no randomized data which characterizes these vaccines' efficacies in populations receiving immunosuppressive anti-cancer therapy.
The prioritization of high first dose uptake, extended dosing intervals in the United Kingdom to a maximum of 12 weeks rather than 3-6 weeks as recommended by the vaccine manufacturers. 4,6 The effect of this off-label dosing in cancer patients is unclear. Worldwide, gastrointestinal malignancies including colorectal, oesophagogastric, and hepato pancreatic and biliary cancers are a leading cause of cancerrelated mortality. 7 Cohort studies to date have reported seroconversion following two doses of mRNA SARS-CoV-2 vaccines in patients with cancer; however, the magnitude of serological responses was lower compared with healthy control groups. [8][9][10] To date, there is a paucity of data reporting the immunogenicity of SARS-CoV-2 vaccines, specifically in patients with gastrointestinal malignancies. To address these concerns, we conducted the CARDS (Cancer: Rapid Diagnostics and Immune assessment for SARS-CoV-2) study to assess the immune status of SARS-CoV-2 immunity in gastrointestinal cancer patients who are receiving anti-cancer therapy.

Study Protocol
The study enrolled patients aged ≥18 years with early or advanced/metastatic malignancy receiving or planning to receive radiotherapy, systemic chemotherapy, or targeted therapy. Eligible patients had no symptoms of acute SARS-CoV-2 infection at enrolment. There were no exclusion criteria. Prior to any study specific procedures, all patients provided voluntary written consent. Enrolled patients were scheduled to have blood taken (serum and EDTA whole blood) at baseline (day 0), day 28, day 56, and day 84. In line with hospital SARS-CoV-2 socially distanced infection control measures, blood tests were scheduled at the time of clinical assessment prior to, or at the time of, anticancer therapy administration. As part of the standard of care, patients received either the Pfizer/BioNtech BNT162b2 or the Oxford/AstraZeneca ChAdOx1 vaccine with a maximum interval of 12 weeks between the first and second doses. 6 Patients were advised to receive a vaccination when invited by local authorities on days when concomitant anti-cancer therapy was not administered.
The primary endpoint was the proportion of patients with a positive detection of (i) anti-nucleocapsid antibodies (anti-NP), and (ii) anti-spike antibodies (anti-S) at each sample timepoint (D0, D28, D56, and D84). The secondary endpoints were the proportion of patients with a positive detection of (i) anti-NP, and (ii) anti-S amongst vaccinated and unvaccinated participants at each timepoint.
The CARDS study was approved by the Newcastle and North Tyneside Research Ethics Committee, United Kingdom (20/NE/0139).

Assays
Serum SARS-CoV-2 S1 RBD Spike antibodies (anti-S) were measured using the COV2T assay on an Atellica analyser (Siemens). Index values ≥1.0 were considered positive as per the manufacturer's protocol. Nucleocapsid (anti-N) antibodies were analyzed with the Elecsys SARS-CoV-2 assay on a Cobas analyser (Roche). As specified by the manufacturer, values above a cut-off index (COI) ≥ 1.0 were reported as positive.
In a 96-well white plate (Grenier Bio-One, Germany), patient serum was diluted 1/20 in DMEM/2% fetal calf serum (FCS)/penicillin-streptomycin, in duplicate, and then combined with an equal volume of pseudovirus in DMEM/2% FCS/penicillin-streptomycin (final serum dilution, 1/40) and incubated for 1 h at 37 °C. Following this incubation, 10 000 HEK293T-AT cells were added to each well. Controls: negative control (cells only), positive control (known neutralizing serum, diluted at 1/80), and a maximum luciferase control (pseudovirus with cells, no serum). The plate was then incubated for 48 h at 37 °C 5% CO 2 . At 48 h the supernatant was removed from each well. Bright-Glo™ luciferase substrate solution (Promega, Madison, WI, USA) diluted 1:1 with PBS was added and read on a luminescence reader.
All wells were normalized to the maximum luminescence control and samples that had an average of 50% or more suppression of luminescence (pVNT50) were deemed neutralizing, allowing the capture of a wide range of responses, while reducing false positives. 12,13 For serum samples that were pVNT80 positive at 1 in 40, a further dilution series was carried out. Starting at 1 in 40 and then 2-fold serial dilutions 8 times, in columns, to 1 in 5120 in a 96-well white plate. The dilution at which the sample still achieved pVNT80 was recorded.

Statistical Analysis
All patients who provided at least 1 blood sample were included in the analysis. All analyses were performed in STATA (v17.0), including the calculation of 95% CIs for proportions and the creation of all plots. The CONSORT diagram was created in Microsoft Visio.

Role of the Funding Source
The funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the manuscript. D.L., A.T., M.A., and S.R. had full access to all the data. S.R. had final responsibility for the decision to submit for publication.

Patient Characteristics
Between September 2020 and April 2021 which was predominantly during the peak of the Delta variant of concern, 152 patients undergoing chemotherapy at the Royal Marsden Hospital, London, United Kingdom were recruited to the study. Of these, 17 patients died or withdrew from the study including 2 patients who were replaced prior to the collection of any blood samples. Across all timepoints, 501 blood samples were taken with results available for 496 blood samples ( Fig. 1).

Primary Outcome
Anti-S antibodies were detected at D0, D28, D56, and D84 in 34.9% (95% CI, 27.  Table S1).  The Oncologist, 2023, Vol. 28,No. 1 Unvaccinated Patients SARS-CoV-2 vaccines were available from December 2020. Fifty-one participants did not receive a vaccine dose prior to, or during the study. Five patients (9.8%) had detectable anti-NP and anti-spike antibodies during the course of the study which was due to prior COVID-19 infection (Supplementary Table S2).

Vaccinated Cohort
SARS-CoV-2 vaccines were available in the United Kingdom from December 2020. Ninety-nine patients received at least 1 dose of a vaccine prior to enrolment or during the study. Forty-six patients (46%) received Pfizer BioNtech BNT162b2 and 50 patients (51%) received the Oxford/AstraZeneca ChAdOx1 vaccine whilst in 3 patients (3.0%) the vaccine received was undetermined. The median duration between the first and second dose received was 11.0 weeks (IQR 9.5, 11.7).
Next, we determined neutralizing antibody activity by a pseudovirus assay which relies upon replication-defective viral particles expressing the "wild-type" SARS-CoV-2 Spike protein 11  As part of a sensitivity analysis, we excluded patients with positive anti-NP as the previous infection confers prolonged protective antibody responses. 14 The positivity rates of anti-spike and neutralizing antibodies were similarly high (Supplementary Table S3).
To further assess the longitudinal evolution of anti-S antibody responses following vaccination, we analyzed the vaccine cohort from the date of the first vaccine. Within the first 19 days following the 1st dose of SARS-CoV-2 vaccination, 30.0% (95% CI, 17.9-44.6) had anti-S detected in serum with 40.4% (27.0-54.9) having neutralizing antibodies. By days 20-39 this had increased to 70.2% (56.6-81.6) and 71.9% (58.5-83.0), respectively. Consistent with previous reports following single-dose vaccination, 15 there was a plateau in seropositivity at days 40-59 (64.4%) and neutralizing antibody activity also mirrored this trend (Fig. 2). The majority of patients with previous COVID-19 infection had high anti-S antibody levels and pseudoviral neutralization activity.

Sensitivity/Specificity
To validate the performance of the Atellica COV2T assay in assessing vaccine responses we excluded patients with positive anti-NP antibodies. In comparison to pseudovirus neutralization, the sensitivity, and specificity of anti-S antibody detection were 80.6%, and 95.7%, respectively (D0). Across all time points, the sensitivity and specificity were similarly high (Supplementary Table S4).

Discussion
To our knowledge, this is the largest, prospective study of a cohort of gastrointestinal cancer patients receiving anti-cancer treatment to have characterized the response to SARS-CoV-2 vaccination. We demonstrated patients were able to mount humoral immune responses to SARS-CoV-2 vaccines and that the immunological responses to vaccination were maintained in patients who had received 1 dose of vaccine prior to systemic cancer therapy initiation. Following a second dose of vaccination, anti-S antibody and pseudovirus neutralization positivity were high and in keeping with previous reports in other solid tumor cohorts. 8,10,[16][17][18][19] Two patients with non-Hodgkin lymphoma receiving anti-CD20 therapy did not have anti-S or pseudovirus neutralizing antibodies detectable after 2 doses of vaccination. Previous reports have confirmed even poorer seroconversion rates in patients with hematological malignancies including acute leukaemia, 20 multiple myeloma, 21 chronic lymphocytic leukaemia, 22,23 and lymphomas particularly in patients receiving B-cell depleting therapies such as anti-CD20 therapy. 24 This highlights the need for booster vaccinations and non-pharmacological interventions to  In the United Kingdom, the duration between the first and second doses of vaccination was extended from 3 weeks to 12 weeks after reports of higher vaccine efficacy and to increase vaccine availability to the wider public. 6 After 40-59 days following the first dose, we observed a small drop in detectable anti-S antibodies which was abrogated by the administration of a second vaccine dose. Whilst this observation may provide an argument to shorten the dosing interval, the low number of infections is also evidence of the effectiveness of other measures such as social distancing, hygiene practices, and hospital infection control policies. This study was conducted during the peak of the Delta (B.1.617.2) variant outbreak in the United Kingdom and these non-pharmaceutical interventions are recommended particularly during outbreaks of highly infectious SARS-CoV-2 variants such as Omicron (B.1.1.529).
Our data suggest a decrease in pseudovirus neutralization titers after 20-39 days following the second dose of SARS-CoV-2 vaccination. Due to the length of follow-up in this study and the size of the sample set, we were not able to definitively assess the duration of vaccine immunogenicity or the factors underpinning this observation. Nevertheless, this result is in keeping with recent reports which have confirmed serological responses to SARS-CoV-2 vaccines significantly wane after 6 months in healthy subjects. 14 We validated the use of qualitative anti-S antibody measurement as a surrogate for SARS-CoV-2 immunity. At the manufacturers' recommendation of an anti-S antibody positivity cut-off of >1.00, high rates of sensitivity and specificity were observed when compared with in vitro pseudovirus neutralization. International standardization of anti-S antibody measurements and neutralization assays is underway which will be useful in determining the value of using serum antibodies as a surrogate for vaccine effectiveness. [25][26][27] Our study is not without its limitations. One of the caveats to our study is the inevitable rates of attrition and study compliance which largely occurred due to constrains on hospital visits; hence, selection bias should be considered when interpreting these data. This is particularly pertinent given patients with solid organ malignancies mount lower anti-S antibody titers compared to healthy subjects. Whilst we did not measure the level of SARS-CoV-2 immunity in relation to a control cohort, we  would expect immune responses to be attenuated, given previous reports. 10,28 Our pseudovirus neutralization assays were modeled upon the Wuhan strain SARS-CoV-2 Spike protein, and viral neutralization to other variants of concern (VOC) was not assessed. Whilst recent studies have observed significant immune escape with the Delta and Omicron variants, 29,30 vaccine booster doses based upon the first wave virus are effective against these VOCs. Given the waning in vaccine immunity and the emergence of VOCs, booster doses are now widely recommended. 31 Serological responses are only part of the protective immune response and whilst we did not assess other mechanisms of SARS-CoV-2 cell-mediated immunity measurable in peripheral blood such as T-cell immunity. Previous studies have reported serological assays and virus neutralization are well-correlated following natural infection 25 and correlate with vaccine effectiveness. 32 It is reassuring that previous studies have been in line with our observations and also report that T-cell responses are maintained following SARS-CoV-2 vaccination in patients with solid organ malignancy. 16 In summary, we have demonstrated that in gastrointestinal cancer patients undergoing chemotherapy, recipients of SARS-CoV-2 vaccination are able to mount immunological responses to a primary course of SARS-CoV-2 vaccination. Whilst these data should provide reassurance to patients with cancer and for clinicians when deciding upon cancer treatments during the COVID-19 pandemic, the duration of humoral responses is likely to be limited and booster doses are recommended.