This study showed that disease-free NHL patients, treated with rituximab-containing immunochemotherapy, have a significant lack of humoral response to influenza vaccine. This was consistently detected for each of the three vaccine Ags and persisted long after treatment (50% of patients evaluated received the last chemotherapy >29 mo before the vaccination). To our knowledge, this is the first study assessing the humoral response to influenza vaccination in lymphoma patients in long-standing remission, outside the perichemotherapy period, thereby avoiding the confounding exerted by the immunosuppressive activity of the disease and/or the antiblastic-induced cytopenia.
There are few data on the activity of influenza vaccine in NHL patients treated with rituximab. Although it is generally accepted that patients with cancer undergoing treatment have a lower humoral response to influenza vaccination than do healthy controls (1
), a recent large trial showed that all parameters investigated in NHL patients following vaccination fulfilled CHMP requirements (6
). However, the cohort studied was more heterogeneous than ours, because some of their patients had not been treated, and only five patients had received rituximab (6
). More recently, Ljungman et al. (28
) described a very weak response to one or two doses of influenza vaccination in patients with various hematological malignancies who had recently discontinued treatment. These results were ascribed, in part, to the inclusion of patients treated with mAbs, because extremely insufficient responses were detected in the six patients who received rituximab. In another study, Takata et al. (29
) evaluated seven NHL patients who were recently treated or were being treated with rituximab and CHOP chemotherapy; they reported virtually no Ab response against one of the three virus strains (i.e., A/H3N2). Although the small number of patients in the aforementioned studies prevents more general conclusions, additional evidence on the immunosuppressive effect of rituximab on influenza response is provided by the observation that, in patients affected by rheumatoid arthritis, it reduced the response to influenza vaccination in the peritreatment period (30
). This is not surprising, because in the peritreatment period (e.g., first 6 mo) B cell depletion is massive, and CD19+
B cells are virtually absent in peripheral blood. The present results, obtained prospectively in a relevant and relatively homogeneous cohort of lymphoma patients, confirmed and extended previous preliminary observations from a limited number of patients and raise the concern that this immune impairment is not limited to the peritreatment period but, rather, persists long-term.
In the current study, seven of nine CHMP requirements for influenza vaccine evaluation were not fulfilled, and the European immunogenicity criteria were not met in the investigated patients (23
). This was true even when adopting the CHMP criteria for the elderly, which are obviously less stringent than those for young people. Although most patients showed a postvaccination titer ≥ 40 for A strains, it is difficult to consider our cohort of patients sufficiently protected against influenza infection. Indeed, this protective threshold value has only been validated in young healthy adults (34
); it may not represent a valid surrogate of clinical protection in frail patients (e.g., the elderly) (35
) or in lymphoma survivors in whom the health status is often compromised by several other causes (36
). In this regard, the increased number of ILI cases observed during the epidemic season could be used as a clinical argument to support these considerations, although the unavailability of specimens for virological analysis prevented an etiological assessment. Notably, all four patients who developed ILI had a low serum level of IgM (+/− low serum level of IgG and/or low serum level of IgA).
A poor response to vaccination was observed against B strains in patients, as well as among the healthy controls. This finding is not surprising, because a lower response against type B influenza viruses compared with type A strains was reported in immuno-competent or immunocompromised individuals of different ages (38
After multivariate analyses, low serum IgM or IgA level, fludarabine administration, and multiple lines of treatment were associated with low serological response to influenza vaccination for at least one of the output variables considered (e.g., seroconversion rate, seroprotection rate, and corrected T1
titers). A history of fludarabine administration was the single factor with the greatest impact on the response to vaccination (in terms of number of significant correlations), and it was the sole factor confirmed by the multivariate modeling when the humoral response was evaluated globally (i.e., through the evaluation of the mean corrected T1
titer). The association among fludarabine, low serum Ig levels, and low B cell/CD27+
memory B cell values strongly suggested a possible detrimental effect of this drug on the reconstitution of the immune system. However, it is well known that fludarabine suppresses the T and B cell arm of the immune response (40
). The correlation between IgM (but not IgG or IgA serum levels) and the postvaccination titer (i.e., mean corrected T1
titer) and the association between low IgM serum levels and fludarabine administration are in agreement with the reported high incidence of infections associated with hypogammaglobulinemia (particularly of the IgM class) in NHL patients treated with rituximab-fludarabine regimens (14
). Additionally, because cellular immune response is also correlated with vaccine protection (35
), the evaluation of Ag-specific T cell responses represent another interesting subject that could be addressed in future investigations.
The coadministration of rituximab and high-dose chemotherapy associated with autologous stem cell transplantation results in a high incidence of IgG hypogammaglobulinemia with CD27+
B cell depletion (10
). On the contrary, IgM hypogammaglobulinemia has not been reported frequently as one of the side effects of rituximab. However, it is noteworthy to highlight that IgM serum determinations were rarely (8
) included in large international rituximab trials (8
Although the number of circulating B cells was similar in patients and healthy controls, a decrease in absolute numbers and relative percentages of CD27+
memory B cells was consistently observed in the patient group. CD27+
memory B cells (classical memory cells) represent the large majority of memory B cells (46
) and are considered responsible for the generation of the bulk of anti-influenza Abs (47
). Although delayed recovery of CD27+
memory B cells was reported in patients followed for shorter periods (10
), the striking finding of the current study is that in NHL patients in CR treated with conventional rituximab-containing chemotherapy, the generalized depletion of CD27+
B cells was still detectable and profound >5 y after the last rituximab dose, and it correlated directly with low serum IgM levels and response to influenza vaccine.
The profound depletion in CD27+
B cells (<10% of CD19+
B cells in patients versus ~40% in healthy subjects) likely includes the reduction of circulating IgM+
B cells, which originate in the splenic marginal zone and in other marginal zone-equivalent areas of lymphoid tissues (48
B cells, which represent ~20% of circulating B cells in healthy subjects, have been considered responsible for the secretion of low-affinity natural Abs (49
). Therefore, this provides a plausible explanation for the low serum IgM levels seen in our patients.
The impairment in CD27+ memory B cells may also be contributed to the low Ab HI titers observed. Because of the design of the present work, the issue cannot be addressed in further detail. Although the IgG fraction of anti-hemagglutinin Abs is the largest Ig component 1 mo after influenza vaccination, the intrinsic inability of the HI assays to differentiate the contribution of the different isotypes to Ab titers represents a limitation of the present observation.
In conclusion, disease-free NHL patients treated with rituximab-based chemotherapies, especially fludarabine, have defective responses to influenza vaccination years after treatment. Persistent perturbation of B cell compartments and Ig synthesis due to rituximab-containing regimens was identified as a mechanism contributing to this low humoral response. Although influenza vaccination remains the most relevant preventive measure in this population, it does not seem to elicit sufficient immune responses in many patients. Our results lay the foundation for larger studies to confirm whether these patients are at particular risk for infections. NHL survivors require careful postvaccination surveillance, and alternative prophylactic/therapeutic approaches represent an urgent subject.