10. STATISTICAL ANALYSIS A description of the participant population will be included in a statistical output report, including subgroups of gender, tumor type, treatment, and treatment intent (curative versus non-curative). Participants who are seropositive to SARS -CoV-2 at baseline and participants who have not received at least one dose of the vaccine will be excluded from th e immune respo nse analyses, including the primary analysis. All subjects who received at least one administration of the vaccine will be included in the analyses of AEs , SAEs and AESIs, a s well as irAEs for cohort s B and D. For description of the results of this study, appropriate descriptive statistics will be used, including estimates of variance. For comparisons between patient cohorts and controls, the most robust appropriate statistical test s will be applied, after checking that all assumptions for a specific test are met. We will be fully transparent about the analyses (including scripts for analyses) and will report the testing results of the assumptions. In addition, we will present all individual data points of the primary and secondary study parameters. 10.1 Primar y study parameter The primary study parameter i s the a ntibody based immune response to vaccination against COVID -19 on day 28 after the second vaccination in cancer patients as compared to individuals without cancer . SARS -CoV-2 S-specific serum IgG antibod y concentrations will be measured at the RIVM using a validated fluorescent bead -based multiplex - immunoassay [ 40]. Geometric mean concentrations (GMCs) and 95% confidence intervals (CIs) will be calculated for the SARS -CoV-2 S-protein -specific IgG antibodi es at baseline and day 28 after vaccination for each cohort. Participants will be classified as responders or non -responders. The definition of response is seroconversion defined as presence of SARS -CoV-2 spike S1 -specific IgG antibodies with a threshold f or seropositivity based on Receiver Operator Curve (ROC) analysis and set at 1.04 AU/mL [41]; in individuals without measurable anti -S antibodies at baseline. Concentrations have been interpolated from a reference consisting of pooled sera using a 5 -parameter logistic Version number: 6.0, 27 December 2021 33 of 49 fit that was calibrated against the NIBSC/WHO COVID -19 reference serum 20/136 and expressed as international binding antibody units per ml (BAU/mL). The value of 1.04 AU/m L corresponds to 10 BAU/mL. Participants who are seropositive at baseline will not be included in the analysis of the primary endpoint. The percentage of responders and corresponding 95% CI for each cohort will be calculated. The percentage of responders of each patient cohort will be compared with the percentage responders in the group without cancer , using a modifi ed standard fixed ‐delta test [42]. 10.2 Secondary study parameter s  Incidence and severity of solicited AEs during 7 days after each vaccination is a key secondary endpoint (see Appendix 1) . Frequencies and absolute numbers of mild, moderate and severe solicited AEs per cohort will be listed for the first , second and third vaccination separately.  The numbers, nature and severity of SAEs graded according CTCAEv5.0 will be listed per cohort.  Incidence and nature of newly occurring irAEs grade ≥ 3 in cohort s B and D up to 28 days after the second vaccination is the second safety endpoint. irAEs will be graded according CTCAE v5.0. Frequencies and absolute numbers of all newly occurring irAEs grade ≥ 3 that occur between the first vaccination and 28 days after the second vaccination in cohorts B and D will be listed.  Incidence, nature and severity of AESIs comprise the third safety endpoint. AE SIs will be graded according CTCAE v5.0. Absolute numbers and f requencies of all AESIs will be listed per cohort and subdivided by severity.  Levels of SARS -CoV-2 S-specific IgG antibodies at 28 days after the second vaccination will be compared between each patient cohort and the non -cancer cohort using ANOVA or Kruskal –Wallis one -way analysis of variance , and Welch t- tests or Mann -Whitney U tests, depending on the distribution of the data .  Levels of SARS -CoV-2 S-specific IgG antibodies before the second vaccination and at 6, 11 and 18 months after the second vaccination will be measured to assess early antibody response and longevity. GMCs and 95% CIs will be calculated . The absolute numbers and percentages of responders will be reported for each cohort for each time point. Antibody levels before the second vaccination and at 6, 11 and 18 months will be compared between each patient cohort and the non-cancer cohort using ANOVA or Kruskal –Wallis one -way analysis of variance, and Welch t-tests or Mann -Whitney U tests, depending on the distribution of the data. Version number: 6.0, 27 December 2021 34 of 49  SARS -CoV-2 specific T cell response will be measured at baseline, and at 28 days after the second and third vaccination , and 6 , 11 and 18 months after the second vaccination and expressed as the number of IFN -ɣ producing SARS -CoV2 specific T cells/million PBMC . To assess the contribution of CD8+ T cells in this response, we will asses s the number of IFN -ɣ producing T cells after blocking with an MHC class I antibody. This will be expressed as the number of IFN -ɣ producing CD4+ T cells/ million PBMC s. Subtraction of the number of responding CD4+ T cells from the total number of respondi ng T cells will lead to the number of IFN -γ producing CD8+ T cells/ million PBMC s. Results will be compared between each patient cohort and the cohort of individuals without cancer using ANOVA or Kruskal – Wallis one -way analysis of variance, and Welch t -tests or Mann -Whitney U tests, depending on the distribution of the data. 10.3 Other study parameters  Dynamics of SARS -CoV-2 specific antibody concentrations will be analyzed , e.g. by cal culating geometric mean fold -rise between baseline and post -baseline time points and antibody decay after day 28.  In-depth flow -cytometric analyses for functional and phenotypical characterization of SARS -CoV-2 specific cell ular responses will be performed by assigning proliferative capacity, cytokine production and phenotypical markers (>25 markers in parallel) in a subset of patients. These descriptive study parameters will not be statistically compared between cohorts.  Baselin e (immune) parameters will be related to vaccination response using univariate analysis .  As a functional readout, the neutralizing capacity SARS -CoV-2 antibodies will be measured at baseline, 28 days and 6 , 11 and 18 months after the second vaccination . Titers will be expressed as GMCs with 95% CIs for each cohort, for each time point .  To assess induction of a mucosal antibody response, m ucosal lining fluid samples will be collected for measurement of SARS -CoV-2-specific antibody concentrations with multiplex immunoassay at baseline and at 28 days after the second vaccination . Additional analyses e.g. n eutralizing capacity of mucosal antibodies can be performed.  Mucosal SARS -CoV-2-specific antibody response will be correlated with serum SARS -CoV-2-specific antibody response. Version number: 6.0, 27 December 2021 35 of 49  Information on incidence of SARS -CoV-2 infection , outcome of COVID -19 during 18 months after the second vaccination will be collected. The number of participants tested, the number of SARS -CoV-2 tests and test results will be reported. For participants with a positive test, information about severity will be presented including hospital admissions, use of oxygen, intensive care admission and mechanical ventilation.  Levels of SARS -CoV-2 S-specific IgG antibodies before the third vaccination , and at day 28 after the third vaccination will be measured to assess antibody response in participants with an inadequate response to two mRNA -1273 vaccinations .  Safety of a third mRNA -1273 vaccination in participants with an inadequate response to two mRNA -1273 vaccinations as mentioned under 10.2 . 10.4 Interim analysis (if applicable) Not applicable.