Effect of dimethyl fumarate on lymphocytes in RRMS: Implications for clinical practice

Abstract

Objective: To assess functional changes in lymphocyte repertoire and subsequent clinical implications during delayed-release dimethyl fumarate (DMF) treatment in patients with multiple sclerosis.

Methods: Using peripheral blood from several clinical trials of DMF, immune cell subsets were quantified using flow cytometry. For some patients, lymphocyte counts were assessed after DMF discontinuation. Incidence of adverse events, including serious and opportunistic infections, was assessed.

Results: In DMF-treated patients, absolute lymphocyte counts (ALCs) demonstrated a pattern of decline followed by stabilization, which also was reflected in the global reduction in numbers of circulating functional lymphocyte subsets. The relative frequencies of circulating memory T- and B-cell populations declined and naive cells increased. No increased incidence of serious infection or malignancy was observed for patients treated with DMF, even when stratified by ALC or T-cell subset frequencies. For patients who discontinued DMF due to lymphopenia, ALCs increased after DMF discontinuation; recovery time varied by ALC level at discontinuation. T-cell subsets closely correlated with ALCs in both longitudinal and cross-sectional analyses.

Conclusions: DMF shifted the immunophenotype of circulating lymphocyte subsets. ALCs were closely correlated with CD4⁺ and CD8⁺ T-cell counts, indicating that lymphocyte subset monitoring is not required for safety vigilance. No increased risk of serious infection was observed in patients with low T-cell subset counts. Monitoring ALC remains the most effective way of identifying patients at risk of subsequently developing prolonged moderate to severe lymphopenia, a risk factor for progressive multifocal leukoencephalopathy in DMF-treated patients.

Trial registration numbers: EUDRA CT 2015-001973-42, NCT00168701, NCT00420212, NCT00451451, and NCT00835770.

Figure 1. Absolute lymphocyte counts (ALCs) over 8 years of dimethyl fumarate (DMF) treatment and lymphocyte subsets by ALC categories

(A) ALCs over 8 years of DMF treatment. Mean (± SE) and 95% confidence intervals (CIs) are presented (n = 2,470). Lower limit of normal (LLN) (0.91 × 10⁹/L) is signified by the dotted line. ^aData not presented for <10 patients. (B) Lymphocyte subsets by ALC categories CD4⁺ LLN = 0.2 × 10⁹/L; CD8⁺ LLN = 0.1 × 10⁹/L. Median (values inside the bars) and 95% CIs are presented. Data were stratified by all ALCs collected during the study (ALC 0.5 to <0.8 × 10⁹/L for ≥6 months [moderate, prolonged lymphopenia]; ALC <0.5 × 10⁹/L for ≥6 months [severe, prolonged lymphopenia]; ALC always ≥0.91 × 10⁹/L [always ≥ LLN]). Patients were categorized as having moderate to severe prolonged lymphopenia if they met these criteria at any time during the study. NK = natural killer.

Figure 2. Immune cell repertoire changes over 24 weeks of dimethyl fumarate (DMF) treatment in PROCLAIM

For each box plot, the top and bottom box edges correspond to the first and third quartiles, n = 163. The black line inside the box represents the median. The top and bottom whisker lines mark the maximum and minimum values of the dataset, respectively. Dotted lines represent lower limit of normal (LLN) (for those subsets with established cutoff values). Absolute lymphocyte count (ALC) LLN = 0.91 × 10⁹/L; CD4⁺ LLN = 0.2 × 10⁹/L; CD8⁺ LLN = 0.1 × 10⁹/L.^, the corresponding p values were calculated using Wilcoxon signed-rank test. Significant p values are marked with asterisks (*p < 0.05 vs baseline; **p < 0.01 vs baseline; ***p < 0.001 vs baseline; ****p < 0.0001 vs baseline). NK = natural killer.

Figure 3. Absolute count median percentage change of immune subsets from baseline to week 24 in PROCLAIM

For each box plot, the top and bottom box edges correspond to Q1 and Q3. The black line inside the box represents the median. The corresponding p values were calculated using Wilcoxon signed-rank test. Significant p values are marked with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Ig = immunoglobulin; NK = natural killer; Q = quartile; Th = T helper.

Figure 4. Proportions of T-cell subsets during early (PROCLAIM) and long-term (ENDORSE integrated analysis) dimethyl fumarate (DMF) treatment

PROCLAIM (n = 163) and the BG00012 Monotherapy Safety and Efficacy Extension Study in Multiple Sclerosis (ENDORSE) integrated analysis (n = 694) are shown. For each box plot, the top and bottom box edges correspond to the first and third quartiles. The black line inside the box represents the median. The top and bottom whisker lines mark the maximum and minimum values of the dataset, respectively. For PROCLAIM (shaded white), the box plots represent the longitudinal trajectory in lymphocyte subsets during early treatment (from baseline [BL] to week 24 [W24]). For the ENDORSE integrated analysis (shaded gray), the box plots represent the cross-sectional observations in lymphocyte subsets in patients who had been exposed to DMF for ∼5 years, stratified by absolute lymphocyte count (ALC) category. For the ENDORSE integrated analysis, data were stratified by ALC categories (ALC always ≥0.91 × 10⁹/L [≥lower limit of normal (LLN)]; ALC <0.8–0.5 × 10⁹/L for ≥6 months [moderate, prolonged lymphopenia, <0.8–0.5]; ALC <0.5 × 10⁹/L for ≥6 months [severe, prolonged lymphopenia, <0.5]). Patients were categorized as having moderate to severe prolonged lymphopenia if they met these criteria at any time during the study. p values were calculated using Wilcoxon signed-rank test, compared the absolute change in relative proportion of lymphocyte subsets at week 24 with baseline for PROCLAIM, were calculated using a pairwise 2-sample Wilcoxon test, and compared the absolute change in relative proportion of lymphocyte subsets for moderate and severe, prolonged lymphopenia compared with ALCs ≥ LLN for the BG00012 Monotherapy Safety and Efficacy Extension Study in Multiple Sclerosis (ENDORSE) integrated analysis. Significant p values are marked with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) vs baseline for PROCLAIM week 24 or vs ≥ LLN for ENDORSE integrated analysis ALC categories. (A) Naive CD4⁺ T cells (CD4⁺/CD45RA⁺/CCR7⁺), effector CD4⁺ T cells (CD4⁺/CD45RA⁺/CCR7⁻), central memory CD4⁺ T cells (CD4⁺/CD45RA⁻/CCR7⁺), effector memory CD4⁺ T cells (CD4⁺/CD45RA⁻/CCR7⁻), and activated CD4⁺ T cells (CD4⁺/CD38⁺/HLA^-DR⁺). (B) Naive CD8⁺ T cells (CD8⁺/CD45RA⁺/CCR7⁺), effector CD8⁺ T cells (CD8⁺/CD45RA⁺/CCR7⁻), central memory CD8⁺ T cells (CD8⁺/CD45RA⁻/CCR7⁺), effector memory CD8⁺ T cells (CD8⁺/CD45RA⁻/CCR7⁻), and activated CD8⁺ T cells (CD8⁺/CD38⁺/HLA^-DR⁺). (C) Naive regulatory T cells (CD4⁺/CD25⁺/CD127^low/-/CD45RO⁻), effector regulatory T cells (CD4⁺/CD25⁺/CD127^low/CD45RO⁺), Th1 phenotype (CD4⁺/CXCR3⁺/CCR6⁻), Th2-enriched phenotype (CD4⁺/CXCR3⁻/CCR6⁻), and Th17 phenotype (CD4⁺/CXCR3⁻/CCR6⁺). Th = T helper.

Figure 5. Proportions of immune cell subsets during early (PROCLAIM) and long-term (ENDORSE integrated analysis) dimethyl fumarate (DMF) treatment

PROCLAIM (n = 163) and the BG00012 Monotherapy Safety and Efficacy Extension Study in Multiple Sclerosis (ENDORSE) integrated analysis (n = 694) are shown. For each box plot, the top and bottom box edges correspond to the first and third quartiles. The black line inside the box represents the median. The top and bottom whisker lines mark the maximum and minimum values of the dataset, respectively. For PROCLAIM (shaded white), the box plots represent the longitudinal trajectory in lymphocyte subsets during early treatment (from baseline [BL] to week 24 [W24]). For the ENDORSE integrated analysis (shaded gray), the box plots represent the cross-sectional observations in lymphocyte subsets in patients who had been exposed to DMF for ∼5 years, stratified by absolute lymphocyte count (ALC) category. For the ENDORSE integrated analysis, data were stratified by ALC categories (ALC always ≥0.91 × 10⁹/L [≥lower limit of normal (LLN)]; ALC <0.8–0.5 × 10⁹/L for ≥6 months [moderate, prolonged lymphopenia, <0.8–0.5]; ALC <0.5 × 10⁹/L for ≥6 months [severe, prolonged lymphopenia, <0.5]). Patients were categorized as having moderate to severe, prolonged lymphopenia if they met these criteria at any time during the study. p values were calculated using Wilcoxon signed-rank test, compared the absolute change in relative proportion of lymphocyte subsets at week 24 with baseline for PROCLAIM, were calculated using a pairwise 2-sample Wilcoxon test, and compared the absolute change in relative proportion of lymphocyte subsets for moderate and severe, prolonged lymphopenia compared with ALCs ≥ LLN for the ENDORSE integrated analysis. Significant p values are marked with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) vs baseline for PROCLAIM week 24 or vs ≥LLN for ENDORSE integrated analysis ALC categories. (A) Transitional B cells (CD19⁺/CD24^hi/CD38^hi), naive B cells (CD19⁺/CD27⁻/IgD⁺), IgD⁺ memory B cells (non-class switched) (CD19⁺/CD27⁺/IgD⁺), IgD⁻ memory B cells (class switched) (CD19⁺/CD27⁺/IgD⁻), plasmablasts (CD19⁺/CD27⁺⁺/CD38⁺⁺), and CD138⁺ plasma cells (CD19⁺/CD27⁺⁺/CD38⁺⁺/CD138⁺). (B) CD4⁺ T cells (CD3⁺/CD4⁺), CD8⁺ T cells (CD3⁺/CD8⁺), B cells (CD19⁺), CD56^bright natural killer (NK) cells (CD19⁻/CD3⁻/CD16⁺/CD56^hi), and CD56^dim NK cells (CD19⁻/CD3⁻/CD16⁺/CD56^low). Ig = immunoglobulin.