Sgherza

Lymphopenia (particularly low CD19+ B-lymphocyte count) and current treatment with either anti-CD38 or anti-BCMA monoclonal antibodies (MoAbs) have been reported to significantly correlate with poor antibody response to conventional doses of anti-SARS-CoV-2 vaccines in patients with multiple myeloma (MM).[1-3] Notably, “booster” doses have been shown to enhance the humoral response of these patients.[4-7] We recently reported the greatly improved clinical outcome of breakthrough SARS-CoV-2 infection in MM patients who had received three or more doses of anti-SARS-CoV-2 vaccines during the different phases of the pandemic COVID-19 including the most recent viral variants of concern (VOCs);[8,9] most of the tested patients had developed an adequate antibody response (anti-spike IgG) to the virus.[9] Due to the scarcity of data about the role of different lymphocyte subsets in this specific population of patients, in the present study, we aimed to evaluate a possible relationship between antibody response after SARS-CoV-2 infection in “booster” vaccinated (at least 3 doses) MM patients and main circulating lymphocyte subpopulations. We also investigated the possible correlation between antibody titer and current treatments, including anti-CD38 MoAbs (daratumumab and isatuximab), in the same patient population. Sixty-two MM patients with breakthrough SARS-CoV-2 infection (men, 58.1%; median age, 65.5 years) followed at our Institution were included in this study between January 2022 and April 2023, when prevailing VOCs were Omicron BA.1, BA.2 and BA.5. Their main baseline characteristics of are listed in Table 1. All patients had been previously vaccinated against SARS-CoV-2 infection with at least three doses. Acquisition of informed consent and collection of serum samples were performed at the first outpatient visit after a median number of 22 days (range: 9-162) from SARS-CoV-2 infection. Determination of anti-spike IgG antibodies was performed using the SARS-CoV-2 IgG II Quant ABBOTT assay, an automated, two-step immunoassay (Chemiluminescent Microparticle ImmunoAssay technology) for the qualitative and quantitative determination of immunoglobulin class G (IgG) antibodies, including neutralizing antibodies to the receptor binding domain of the S1 subunit of the spike protein of SARS-CoV-2 in human serum and plasma. It utilizes a four Parameter Logistic Curve fit data reduction method (4PLC, Y-weighted) to generate a calibration and results. The chemiluminescent reaction is measured as a relative light unit (RLU). There is a direct relationship between the amount of IgG antibodies to SARS-CoV-2 in the sample and the RLU detected by the system optic. Results were reported as arbitrary units (AU), with a positivity cut-off of ≥ 50 AU/mL as an arbitrary threshold for “adequate” response. Flow cytometric analyses were performed for assessment of the patients' lymphocyte status. Briefly, 50 μl of EDTA whole blood was stained with 20 μl of BD Multitest™ CD3/CD16+CD56/CD45/CD4/ CD19/CD8 (FITC-labeled CD3, clone SK7; PE-labeled CD16, clone B73.1, and CD56, clone NCAM 16.2; PerCP-labeled CD45, clone 2D1 (HLe-1); PE-Cy7-labeled CD4, clone SK3; APC-labeled CD19, clone SJ25C1; and APC-Cy7-labeled CD8, clone SK1) in BD Trucount tubes after 15 minutes in the dark at room temperature 450 µL of lysing solution were added to the tube. After 10-15 minutes, samples were analyzed on the BD FACSCanto II flow cytometer. Absolute counts of T cell subsets, B cells, and NK cells were determined by the software BD FACSCanto™. Correlation between different lymphocyte subpopulations and anti-SARS-CoV-2 antibody titers was investigated using Spearman’s Rho criterion, while comparisons between groups were performed by the Mann–Whitney U test. Statistical analyses were carried out using Jamovi (version 2.4.7) and GraphPad Prism (version 8.3.0). The favorable clinical outcome of breakthrough SARS-CoV-2 infection in this cohort of patients has been previously reported;[9] in particular, only 4 hospitalizations (6.4%) were observed, but none in an intensive cure unit. After a median number of 22 days (range 9-162) from positive swabs for SARS-CoV-2, almost all patients (60/62, 96.8%) achieved a titer greater than 50 AU/mL. Only two patients showed a lower titer after 5 and 3 vaccine doses, respectively: a 79-year-old female, receiving isatuximab, pomalidomide, and dexamethasone as 4^th line therapy, and an 82-year-old female, receiving elotuzumab, pomalidomide, and dexamethasone as 3^rd line treatment. At the time of SARS-CoV-2 infection, these patients were respectively in partial response and very good partial response, according to the International Myeloma Working Group (IMWG) criteria. Notably, both these patients showed a low count of CD19+ B-lymphocytes and the concomitant use of pomalidomide. Regarding the antibody response according to the absolute count of CD19+ B-Lymphocytes, the presence of a direct correlation between the two variables (Spearman: 0.417; p= 0.007) (Figure 1A), and a significant difference according to the median value used as a cut-off level (Figure 2A) were observed. By contrast, assessing the impact of the absolute count of other lymphocyte populations on the development of antibody titer, no correlation was found (CD4+: Spearman: -0.010, p = 0.950; CD8+: Spearman: -0.108, p = 0.506; CD16+CD56+: Spearman: 0.098, p = 0.547) (Figure 1, B-D). Likewise, no statistically significant differences in terms of antibody titer emerged comparing patients with lower versus higher median CD4, CD8, and CD16/CD56 positive lymphocyte absolute values (Figure 2, B-D). Finally, evaluating the antibody response according to current treatment with anti-CD38 Mo-abs, no statistically significant correlation was identified between 31 patients undergoing these treatments and 31 patients who did not (Figure 2E). Overall, our study suggests that, in patients who have previously received three or more doses of anti-SARS-CoV-2 vaccines, the absolute number of CD19+ B cells may marginally reduce the production of specific antibodies after breakthrough SARS-CoV-2 infections without significantly decreasing; however, the percentage of patients with potentially “protective” titers. In this setting, the absolute number of T and NK populations, as well as the use of anti-CD38 antibodies for the treatment of MM, did not show significant effects on humoral response to viral infection. Curiously, the only two patients with suboptimal humoral response after breakthrough SARS-CoV-2 infection were both receiving pomalidomide; the possible detrimental effect of this drug on antibody production would warrant further investigation. However, evaluating the antibody response according to current treatment with IMIDs, no statistically significant correlation was identified between 48 patients undergoing these treatments and 14 patients who did not (Figure 2F). The study has several limitations, particularly the limited number of patients tested, the lack of information about serological response and lymphocyte counts before infection, the heterogeneous timing of blood collection, the different types of vaccine employed, and the lack of a control group. Furthermore, the important role of specific functional aspects of T and NK-cell responses to breakthrough SARS-CoV-2 infection in fully vaccinated MM patients[10-12] was not investigated. Notwithstanding, our observation is in line with the generally favorable clinical outcome of COVID-19 we observed in these patients and would seem to reflect the independence of clinical and serological response upon quantitative amounts of different lymphocyte sub-populations present at the time of viral infection, including patients receiving anti-CD38 therapies after booster vaccinations and infected by novel SARS-CoV-2 VOCs that represent the current epidemiological scenario.

Table 1. Clinical and laboratory characteristics of MM patients with breakthrough SARS-CoV-2 Infection after at least three anti-SARS-CoV-2 vaccine doses.

Figure 1. Correlation between antibody titer and absolute count of (A) CD19+B-lymphocytes, (B) CD4+T-lymphocytes, (C) CD8+T-lymphocytes, (D) and CD16+CD56+ NK-lymphocytes.

Figure 2. Comparison of antibody titer according to the median (lower vs higher) absolute count of (A) CD19+B-lymphocytes, (B) CD4+T-lymphocytes, (C) CD8+T-lymphocytes, (D) CD16+CD56+ NK-lymphocytes and (E) the use of anti-CD38 MAbs (yes vs. no) or (F) IMIDs (yes vs. no).

Antibody Response to Breakthrough SARS-CoV-2 Infection in “Booster” Vaccinated Patients with Multiple Myeloma According to B/T/NK Lymphocyte Absolute Counts and anti-CD38 Treatments