The immune system plays a crucial role in inhibiting the pathogenesis and development of malignant neoplasms. In this regard, clinical research focused on the effect of the profile of immune system is performed among patients with hematologic malignancies and those with solid tumors. For example, absolute lymphocyte counts (ALCs), a simple laboratory parameter that is routinely evaluated in the clinical setting, has been found to be associated with treatment outcomes in patients with diffuse large B cell lymphoma, follicular lymphoma, or acute leukemias (1-3). Functional analysis of the immune system focusing on natural killer (NK) cells, regulatory T cells (Tregs), or T cells in peripheral blood has revealed that enhanced or limited immune function could modify treatment outcomes; increased number of NK cells (particularly mature NK cells), helper T cells, and depleted Tregs can positively affect clinical outcomes (4-10).

Multiple myeloma (MM) is a clonal hematologic malignant neoplasm characterized by the accumulation of abnormal plasma cells in the bone marrow. Over the years, the prognosis of patients with MM has improved owing to the introduction of novel agents, including proteasome inhibitors (bortezomib, carfilzomib, and ixazomib), immunomodulatory drugs (IMiDs; thalidomide, lenalidomide, and pomalidomide), and antibodies (elotuzumab and daratumumab) (11,12). In particular, IMiDs and proteasome inhibitors are principal agents for MM therapy. IMiDs are known to enhance the immune system by NK cell expansion and activation, regulatory T cell (Treg) inhibition, and/or restoration of T cell function in in vitro studies (13-15). Among them, lenalidomide has widely been used in combination with dexamethasone with or without other agents (proteasome inhibitors, antibodies, or cytotoxic agents); the efficacy of the combination therapy is well demonstrated by large-scale studies, regardless of initial therapy or refractory/relapsed status (16-22). Although lenalidomide is considered to exert dual antitumor effects, including the activation of normal immune system through the activation of cytotoxic lymphocytes and the proteasome-dependent degradation of transcription factors such as IKZF1 and IKZF3 in myeloma cells (13-15), there is no clinical evidence that directly proves the association between changes in the profile of immune system and treatment efficacy in patients with MM treated with this agent. Therefore, the clinical significance of changes in the immune system after treatment with lenalidomide requires evaluation.

In this study, we assessed the prognosis of patients with RRMM by focusing on absolute lymphocyte counts before and after starting lenalidomide therapy. This study highlights the influence of lymphocyte counts after lenalidomide initiation on outcomes of patients with RRMM.

Patients. We reviewed the medical records of patients with MM in the database of our institute (Nihon University Itabashi Hospital, Tokyo, Japan). Patients with RRMM who were treated with a doublet regimen comprising lenalidomide and dexamethasone (Ld) were included in the study. Our institutional practice with respect to the treatment of MM involves introducing lenalidomide as a subsequent therapy after bortezomib-based therapy, cytotoxic agents, and/or autologous peripheral blood stem cell transplantation. Ld was continued every 28 days without withdrawal until disease progression unless any severe adverse event was observed. Lenalidomide (25 mg/day) was administered for 21 days and dexamethasone (20 mg) was orally administered on days 1, 8, 15, and 22 of a 28-day cycle. The doses of both drugs were reduced at the discretion of the treating physician. Patients who were treated with lenalidomide-containing triplet regimen were excluded from this study because combining agents such as elotuzumab, ixazomib, bortezomib, or daratumumab can influence both the therapeutic effect and the profile of the immune system. Data on ALCs were obtained from routine complete blood count before and at 1, 2, and 3 months after Ld initiation. This retrospective study was approved by the Institutional Review Board of Nihon University Itabashi Hospital (Tokyo, Japan) who provided all necessary ethical permissions (identifier RK-180710-28).

Statistical analysis. The non-parametric Kruskal–Wallis test was used to evaluate associations between continuous variables. Time to next treatment (TNT) was defined as the time between the date of Ld initiation and the date of subsequent treatment initiation, death by any cause, or date of the last follow-up. Overall survival (OS) was defined as the time between the date of Ld initiation and the date of death by any cause or last follow-up. TNT and OS were analyzed using the Kaplan–Meier method and log-rank testing was used for between-group comparisons. Factors that could potentially influence treatment outcomes were analyzed using the Cox proportional hazard regression model. A p-value of less than 0.05 was considered to be statistically significant. Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) (23).

Patient characteristics. Among the patients in our database, 72 patients with RRMM were treated with a doublet Ld regimen between September 2010 and April 2018. Of these 72 patients, 72, 69, 54, and 53 were available for determining ALCs before treatment and at 1, 2, and 3 months after Ld initiation, respectively. Table I shows the characteristics of patients enrolled in the current study. The median age at Ld initiation was 71 years (range=37-82 years). Out of 72 patients, 35 (48.6%) were male and 37 (51.4%) were female. The most common monoclonal protein was the IgG type (n=48, 66.7%), followed by IgA type (n=15, 20.8%), Bence Jones protein type (n=8, 11.1%), and non-secretory type (n=1, 1.4%). Thirty-six patients had undergone 2-5 lines of regimens prior to Ld therapy.

Changes in ALCs and treatment outcomes. The median count of ALCs at Ld initiation was 1,131/μl and that after 1, 2, and 3 months of Ld initiation was 1,059, 1,222, and 1,162/μl, respectively (Figure 1). The number of ALCs showed no significant changes with the course of Ld treatment during the first 3 months. With respect to patient prognosis, there were no differences in both TNT and OS depending on whether the ALCs at Ld initiation were greater than the median (Figure 2A and B). However, TNT and OS in patients with higher ALCs at 1 or 2 months after Ld initiation were slightly better than those with lower ALCs, although the differences were not statistically significant (Figure 3A and B, Figure 4A and B). Patients with higher ALCs at 3 months of Ld initiation showed longer TNT compared to those with lower ALCs at 3 months (Figure 5A). Furthermore, patients with higher ALCs at 3 months of Ld initiation were also closely associated with better OS (Figure 5B). Multivariate analysis of TNT revealed that ALCs higher than the median at 3 months of Ld initiation was an independent favorable prognostic factor for TNT as well as treatment response as indicated by the achievement of partial response (PR). In addition, prior history of thalidomide therapy, which mainly indicates thalidomide-resistance, was an independent adverse prognostic factor for TNT after Ld initiation (Table II). However, ALC higher than the median at 3 months of Ld initiation was not an independent prognostic factor in multivariate analysis of OS (Table III).

In this study, we assessed the prognosis of patients with RRMM by focusing on ALCs before and after starting lenalidomide therapy. We found that a higher ALC at 3 months of lenalidomide initiation was closely related to better outcomes, including TNT and OS, however, ALC before treatment initiation was not associated with better outcomes. Multivariate analysis revealed that higher ALCs 3 months after initiating Ld therapy was an independent favorable prognostic factor for TNT as well as good treatment response as indicated by the achievement at least of PR (≥50% reduction of serum M-protein). This suggests that changes in the profile of the immune system after Ld therapy could favorably affect tumor regression and/or prevent disease recurrence. To our knowledge, this is the first study to evaluate the prognostic value of ALCs in RRMM treated with Ld.

Previous studies have demonstrated the significance of the profile of the immune system on the prognosis of patients with MM treated with bortezomib (24,25). Songet et al. (24) have shown that decreased lower lymphocyte counts were closely associated with shorter progression-free survival in RRMM patients treated with bortezomib plus dexamethasone. Similarly, a higher neutrophil-to-lymphocyte ratio has also been shown to adversely affect OS in newly diagnosed transplant-ineligible MM patients treated with bortezomib-based therapy (25). These results are consistent with the findings of our study, showing the clinical significance of the status of the immune system for MM therapy. It should be emphasized that the current study results indicate the importance of activation or expansion of functional immune effector cells or inhibition of Tregs by this agent because the significance of lymphocyte counts was apparent only after initiating Ld therapy. Indeed, the extent of Treg inhibition after bortezomib or lenalidomide therapy is associated with the achievement of at least very good PR (26).

Some in vitro studies have revealed the effect of lenalidomide on the immune system. Lenalidomide has also been reported to enhance the function of helper T cells, cytotoxic T cells, NK cells, and NK/T cells; to promote Th1 cytokine (interferon-γ and IL-2) production; and to inhibit Treg expansion (14,15). These results demonstrate the contribution of the treatment efficacy of lenalidomide via activation of the immune system. However, in vivo studies have resulted in inconsistent or controversial results. Chan et al. (27) have reported that NK/T cell deficit was observed in RRMM patients but not in newly diagnosed MM patients, and the number of NK/T cells and cytokine levels showed no significant alterations during the course of lenalidomide therapy. Clave et al. (28) have shown that lenalidomide impaired T cell reconstitution and increased the absolute number of Tregs in patients after autologous stem cell transplantation. Similarly, an increase in the number of Tregs after induction therapy with Ld has also been reported (29). In contrast, Lioznov et al. (30) have described that lenalidomide increased the number of activated NK and T cells in patients treated with Ld as a salvage regimen after allogeneic stem cell transplantation. In this regard, we hypothesize that the immune system might be differently affected by lenalidomide based on the differences in the clinical setting. In this study, all the included patients were in the refractory or relapsed setting and were treated with a doublet Ld regimen without combining any other anti-myeloma agent. A unified situation is desirable for performing clinical investigations focused on the association between changes in the immune system and patient prognosis. In addition, the current study results encourage further investigation of changes in the profile of the immune system associated with patients’ outcomes.

Our study has several limitations. First, the change in the profile of the immune system was evaluated only by ALCs without evaluating detailed lymphocyte fractions; therefore, the lymphocyte components affected by lenalidomide remain unidentified. Second, the evaluation of treatment efficacy, such as complete response, could not be formally evaluated because of the lack of data on immunofixation due to the unavailability of the method at the time of lenalidomide approval. Therefore, the therapeutic effects were analyzed only on the basis of whether PR was achieved or not. Finally, a further limitation is potential selection bias, as not all patients were treated with a doublet Ld regimen because triplet regimens combined with proteasome inhibitors or antibody agents have recently become a standard of care for patients with RRMM.

In conclusion, the current study suggested that lymphocyte counts after Ld therapy possibly influence treatment outcomes as a result of modulating tumor immunity. Further research is warranted to clarify the association between the effect of the modification of the immune system by lenalidomide and the prognosis for this set of patients.

NI received honoraria and speaker fees from Bristol-Myers Squibb, Celgene K.K., Takeda Pharma Co., Ltd., and Ono Parma Co., Ltd. KM received honoraria and speaker fees from Celgene K.K., Takeda Pharma Co., Ltd., and Ono Parma Co., Ltd. HT, MN, and TH reports honoraria and speaker fees from Bristol-Myers Squibb. YH received speaker fees and honoraria from Celgene K.K., Janssen Pharmaceutical K.K., Bristol-Myers Squibb, Takeda Pharma Co., Ltd., and Ono Parma Co., Ltd. MT received honoraria from Janssen Pharmaceutical K.K., Bristol-Myers Squibb, Takeda Pharma Co., Ltd., and Ono Parma Co., Ltd., supports for extension lectures from Bristol-Myers Squibb, and scholarship funds from Takeda Pharma Co., Ltd. and Ono Parma Co., Ltd. Also, MT is part of an international clinical trial evaluating an investigational drug that was developed by Bristol-Myers Squibb for the treatment of Sjögren’s syndrome and has cooperative research with Celgene K.K. TN received scholarship funds from Roche Diagnostics K.K. and salary as an instruction doctor of the ethics from the Health Sciences Research Institute, Inc. The remaining co-authors declare no competing financial interests.

MN and NI contributed equally to this study. MN, TI, and NI analyzed and presented results. YH and MT assisted in the interpretation of the results with MN and NI; MN, NI, YH, and MT designed the research and wrote the manuscript. MN, NI, KM, YU, HT, TH, KI, TK, KK, and YH collected patient data. All Authors read and approved the final manuscript.

The Authors thank Ms. Sachiyo Mazume and Ms. Miharu Watanabe for collecting the patients’ data to make this study possible.