Initial lymphocyte count and low BMI
may affect fingolimod-induced
Clemens Warnke, MD*
Thomas Dehmel, MSc*
Ryan Ramanujam, PhD
Carolina Holmen, PhD
Nina Nordin, BSc
Kathleen Wolfram, MSc
Verena I. Leussink, MD
Hans-Peter Hartung, MD
Tomas Olsson, MD, PhD
Bernd C. Kieseier, MD
Correspondence to
Dr. Kieseier:
bernd.kieseier@uni-duesseldorf.de
lymphopenia
ABSTRACT
Objective: To assess whether pretreatment-lymphocyte counts, treatment before fingolimod, age,
sex, or body mass index (BMI) affects the risk of fingolimod-induced lymphopenia in patients with
relapsing-remitting multiple sclerosis (RRMS).
Methods: Data were obtained from a German multicenter, single-arm, open-label study of patients
with RRMS treated with fingolimod, and findings were validated in an independent Swedish
national pharmacovigilance study.
Results: Four hundred eighteen patients with RRMS from Germany and 438 patients from
Sweden were included. A nadir #0.2 3 109 lymphocytes/L was reached in 15% (95% confidence
interval [CI] 12%–17%) of all 856 patients. Patients with lower starting lymphocyte counts
(below 1.6 3 109/L) and patients with BMI lower than 18.5 kg/m2 (women only) were at higher
risk of developing lymphopenia with values #0.2 3 109/L in the combined analysis, increasing the
risk in these subgroups to 26% (95% CI 20%–31%) or 46% (95% CI 23%–71%), respectively.
In the German cohort, infection rates were similar in patients who developed severe lymphopenia
and those who did not.
Conclusions: Our findings suggest that patients with low baseline lymphocyte counts and under-
weight women in which fingolimod treatment will be initiated should possibly be monitored more
closely. Neurology® 2014;83:2153–2157
GLOSSARY
BMI 5 body mass index; CI 5 confidence interval; MS 5 multiple sclerosis; OR 5 odds ratio; RRMS 5 relapsing-remitting
multiple sclerosis.
Fingolimod is a sphingosine-1-phosphate receptor agonist, approved for patients with relapsing-
remitting multiple sclerosis (RRMS). The clinical effect has been attributed to its capacity to
sequester circulating lymphocytes into secondary lymphoid tissues.1 In pivotal studies, fingoli-
mod treatment reduced circulating lymphocytes to a mean of about 0.5 3 109/L.2–4 Lympho-
cyte counts below 0.2 3 109/L were defined as criterion for therapy discontinuation, reached in
about 20% of patients receiving fingolimod 0.5 mg/d on any single occasion during the studies.4
It remains unclear whether pretreatment-lymphocyte counts, treatment before fingolimod,
age, sex, or body mass index (BMI) affects the risk of fingolimod-induced lymphopenia. We
addressed this in a large German MS patient exploratory cohort with samples available before
and during therapy. Findings were validated in a second, independent patient cohort from
the Swedish nationwide pharmacovigilance study for fingolimod.
METHODS Patients. Exploratory cohort. This was a multicenter, single-arm, open-label study of patients with RRMS receiving
0.5 mg fingolimod once daily in Germany. Peripheral venous blood was sampled immediately before (baseline), and 1, 4, and 6 months
after treatment initiation.
Validation cohort. Immunomodulation and Multiple Sclerosis Epidemiology II Study: this is a Swedish nationwide pharmacoe-
pidemiologic and genetic study focused on long-term safety and efficacy of fingolimod,5 which included a total of 1,123 patients with
MS treated with fingolimod (as of June 2014). Patients with lymphocyte counts at baseline and at any time point during therapy with
fingolimod were included.
*These authors contributed equally to this work.
From the Department of Neurology (C.W., T.D., K.W., V.I.L., H.-P.H., B.C.K.), Medical Faculty, Heinrich-Heine University, Düsseldorf,
Germany; and Department of Clinical Neuroscience (R.R., C.H., N.N., T.O.), Karolinska Institute, Stockholm, Sweden.
Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
© 2014 American Academy of Neurology 2153
 Table 1 Patient characteristics

BMI, Baseline lymphocytes,

Basic characteristics Age, y Sex, n (%) female kg/m2 3109/L DMT before fingolimod, no. of patients (%)

German exploratory 39 6 9.5 286 (68.4) 25.4 (5.0) 1.9 6 0.6 IFN: 196 (47); GA: 85 (20); NAT: 62 (15); IS: 6 (1);
cohort (n 5 418) others/none: 69 (17)

Swedish validation 39 6 9.3 295 (67.3) 24.3 (4.5) 2.3 6 0.9 IFN: 131 (30); GA: 39 (9); NAT: 153 (35);
cohort (n 5 438) others/none: 115 (26)

Abbreviations: BMI 5 body mass index; DMT 5 disease-modifying therapy; GA 5 glatiramer acetate; IFN 5 interferon-b; IS 5 immunosuppressive therapy,
azathioprine (n 5 2) or mitoxantrone (n 5 4); NAT 5 natalizumab.
Data are mean 6 SD unless otherwise indicated.

Lymphocyte counts. In the German cohort, all samples were
shipped to one central laboratory within 12 hours at 4°C and total
lymphocyte counts were obtained by automated flow cytometer anal-
ysis in the same machine (Sysmex XN-system; Sysmex Corp., Kobe,
Japan). Standard quality measures were implemented for sample
control. For the validation cohort, counts were provided by the
Swedish MS registry.
Standard protocol approvals, registrations, and patient
consents. Appropriate institutional review boards approved
this study; for the German cohort, approval was given by the
Ethikkommission der Heinrich-Heine Universitaet Duesseldorf
(MC-LKP-470); for the validation cohort, by the Karolinska
Institutet regional ethical committee (2011/641-31/4). All
patients gave written informed consent to the scientific use
of samples and data.
Statistical analysis. Statistical analysis was performed in R6 or
GraphPad Prism version 6.0 (La Jolla, CA). Mean 6 SD and
proportions with 95% confidence intervals (CIs, modified Wald

Figure Lymphocyte counts before and during therapy with fingolimod

Lymphocyte counts are shown at baseline, month (m)1, m4, and m6 after treatment initiation with fingolimod in the German
(A) and at baseline, m6, m12, and m24 in the Swedish cohort (B). The x-y diagrams illustrate the correlation of the baseline
lymphocyte count with the lowest count during the study in the German (C) and the Swedish cohort (D).

2154 Neurology 83 December 2, 2014

Table 2 German or Swedish patients with lymphocyte counts at or below 0.2 3 109/L

No./group % (95% CI) OR (95% CI) p Value

German cohort

All patients 66/418 16 (13–20)

Age, y

<30a 14/81 17 (10–27) — —

30–37 18/98 18 (12–27) 1.02 (0.51–2.07) 0.945

38–44 22/112 20 (13–28) 1.26 (0.64–2.50) 0.500

‡45 12/127 9 (5–16) 0.56 (0.26–1.23) 0.152

Sex

Female 49/286 17 (13–22) 1.18 (0.67–2.10) 0.564

Male 17/132 13 (8–20) — —

BMI, kg/m 2

<18.5 5/11 45 (21–72) 3.28 (1.18–9.12) 0.023b

18.5–25c 37/230 16 (12–21) — —

25–30 15/124 12 (7–19) 0.77 (0.43–1.39) 0.392

‡31 9/53 17 (9–29) 1.02 (0.49–2.09) 0.964

Baseline lymphocytes

<1.6 3 109/L 37/146 25 (19–33) 2.25 (1.37–3.70) 0.001b

‡1.6 3 109/L 29/272 11 (7–15) — —

DMT before fingolimod

IFN 38/196 19 (14–26) 1.14 (0.55–2.33) 0.728

GA 6/85 7 (3–15) 0.37 (0.13–1.01) 0.051

NATc 11/62 18 (10–29) 1.02 (0.50–2.06) 0.955

IS 1/6 17 (1–58) 1.10 (0.14–8.66) 0.931

Others/none 10/69 14 (8–25) 0.79 (0.32–1.94) 0.609

Swedish cohort

All patients 59/438 13 (11–17)

Age, y

<30a 10/72 14 (8–24) — —

30–37 21/124 17 (11–25) 1.08 (0.50–2.32) 0.841

38–44 11/125 9 (5–15) 0.52 (0.22–1.24) 0.140

‡45 17/117 15 (9–22) 0.82 (0.37–1.83) 0.624

Sex

Female 48/295 16 (12–21) 2.56 (1.32–4.98) 0.005b

Male 11/143 8 (4–13) — —

d
BMI, kg/m (total n 5 55 )
2

<18.5 1/2 50 (9–91) — —

18.5–25 3/26 12 (3–30) — —

25–30 3/20 15 (4–37) — —

‡31 2/7 33 (8–65) — —

Baseline lymphocytes

<1.6 3 109/L 25/96 26 (18–37) 2.77 (1.56–4.90) 0.0005b

‡1.6 3 109/L 34/342 10 (7–14) — —

DMT before fingolimod

IFN 26/131 20 (14–28) 0.89 (0.44–1.79) 0.744

GA 2/39 5 (0–18) 0.28 (0.06–1.24) 0.094

Continued
Neurology 83 December 2, 2014 2155
 Table 2 Continued
No./group
c
NAT 18/153
Others/none 13/115
Abbreviations: BMI 5 body mass index; CI 5 confidence interval; DMT 5 disease-modifying therapy; GA 5 glatiramer acetate; IFN 5
interferon-b; IS 5 immunosuppressive therapy, azathioprine (n 5 2) or mitoxantrone (n 5 4); NAT 5 natalizumab; OR 5 odds ratio.
a
Reference in Cox regression, no OR available.
b
Significant values.
c
Obtained using a pruned model because of colinearity.
d
BMI questionnaire data obtained within 5 years of start with fingolimod; Cox regression underpowered because of limited data.
method) were calculated. To assess differences in longitudinal
paired samples, the repeated-measures 1-way analysis of variance
was used with correction for multiple testing. To test for
association of variables to the risk of developing lymphopenia
and to account for incomplete data, Cox regression with
lymphocyte count below 0.2 (exploratory cohort, 3-digit values
behind the comma) or at 0.2 3 109/L (validation cohort, $1 place
after the decimal point) as censoring event was conducted. Initial
lymphocyte count, treatment before fingolimod, age, and BMI
were tested as categorical or continuous covariates.
RESULTS Four hundred eighteen German and 438
Swedish patients with RRMS fulfilled inclusion crite-
ria with availability of lymphocyte counts at baseline
and at any time point during therapy (German
cohort: month [m]1 [n 5 412], m4 [n 5 397], m6
[n 5 402]; Swedish cohort: m1 [n 5 386], m3 [n 5
312], m6 [n 5 317], m9 [n 5 160], m12 [n 5 185],
m15 [n 5 55], m18 [n 5 67], m21 [n 5 33], m24
[n 5 41]) (table 1). In the German and the Swedish
cohorts, on average 2.9 and 3.6 time points per
patient during therapy, respectively, were available.
Compared with pretreatment values (German cohort:
1.89 6 0.63 3 109/L; Swedish cohort: 2.27 6 0.90 3
109/L), mean lymphocyte counts were lower at m1
(German cohort: 0.56 6 0.32 3 109/L; Swedish cohort:
0.63 6 0.31 3 109/L), and remained low throughout
therapy (m6: German cohort: 0.55 6 0.39 3 109/L;
Swedish cohort: 0.51 6 0.27 3 109/L; m24: Swedish
cohort: 0.57 6 0.28 3 109/L) (figure, A and B).
The lymphocyte counts were reduced below
(German cohort) or at 0.2 3 109/L (Swedish cohort)
on at least one of the time points during therapy in 16%
(95% CI 13%–20%) or 13% (95% CI 11%–17%),
respectively (table 2). This nadir was repeatedly (twice
or more) reached in 8 of 418 patients (2% [95% CI
1%–4%]) of the German cohort, and 23 of 438 pa-
tients (5% [95% CI 3%–8%]) of the Swedish cohort.
Cox regression sensitivity analysis demonstrated
increased risk with initial lymphocyte count below
1.6 3 109/L separately in the German and the Swed-
ish cohort (table 2), increasing the estimated propor-
tion of patients with lymphopenia in the combined
dataset to 26% (95% CI 20%–31%; odds ratio [OR]
2.60 [95% CI 1.80–3.76]; p 5 3.4 3 1027).
2156 Neurology 83 December 2, 2014
% (95% CI) OR (95% CI) p Value
12 (7–18) 1.27 (0.08–1.40) 0.440
11 (7–19) 0.56 (0.26–1.19) 0.130
Accordingly, there was a moderate positive correla-
tion for the initial lymphocyte count and the lowest
count during the study, independently observed in
the German and Swedish cohorts (figure, C and D).
Furthermore, BMI ,18.5 kg/m2 (only women in
our study) was associated with a higher risk of
fingolimod-induced lymphopenia below or at 0.2 3
109/L in the German cohort, and a similar direction
in the Swedish cohort (table 2; BMI data available in
only 55 of the Swedish patients), increasing the esti-
mated proportion of patients with lymphopenia in
the pooled cohort to 46% (95% CI 23%–71%;
OR 3.61 [95% CI 1.41–9.27]; p 5 0.007).
Prior therapy with immunosuppressant drugs (mi-
toxantrone or azathioprine) or natalizumab did not
increase the risk of lymphopenia. Pretreatment with
glatiramer acetate showed a “protective” trend in the
German and the Swedish cohort (table 2), decreasing
the proportion of patients with lymphopenia in the
pooled dataset to 6% (95% CI 3%–12%; OR 0.33
[95% CI 0.15–0.73]; p 5 0.006).
Females were at higher risk of lymphopenia at or
below 0.2 3 109/L in the Swedish, and showed a
similar direction in the German cohort (table 2),
increasing the proportion of patients with lymphope-
nia in the pooled cohort to 17% (95% CI 14%–20%;
OR 1.87 [95% CI 1.23–2.86]; p 5 0.004).
Age had no effect on the risk of reaching a nadir
below or at 0.2 3 109 lymphocytes/L. Infections were
reported in the German cohort only. In the 66 pa-
tients who reached a nadir of ,0.2 3 109/L during
the 6-month observational period, 27 (41%, 95% CI
30%–53%) had one or more infections, which did
not differ from the overall infection rate of the total
cohort (37%, 95% CI 33%–42%).
DISCUSSION In the 2 published phase-III clinical
studies, lymphocyte counts dropped to an average of
approximately 0.4 6 0.26 3 109/L in patients
receiving 1.25 mg and 0.5 6 0.31 3 109/L for
those receiving 0.5 mg fingolimod,2,3 independent
of the timing of meals and not influenced by
comedications, such as corticosteroids.7 Even 5.0 mg
did not result in a more pronounced lymphopenia.8
The relevance of the reduced lymphocyte count
remains uncertain, although the absolute lymphocyte
count did not correlate with the rate of infections in
the phase-III studies.4
In our study, 15% of all 856 patients (pooled
German and Swedish dataset) developed lymphopenia
below or at 0.2 3 109/L on at least one of the approx-
imately 3.2 time points/patient studied. Of note, pa-
tients with a low initial lymphocyte count (,1.6 3
109/L) or with a BMI ,18.5 kg/m2 (females only)
exhibited a 2.6- to 3.6-fold increased risk of dropping
below this threshold, elevating the estimated proportion
experiencing the event to approximately 26% or 46%,
respectively. Furthermore, pretreatment with glatiramer
acetate, based on our statistical analysis, suggested a pro-
tective effect in our pooled dataset, with only 6% reach-
ing a lymphocyte nadir of #0.2 3 109/L; however, it
remains questionable to which extend this mathematical
observation mirrors a relevant immunologic effect of
glatiramer acetate.
There are limitations to our study: missing data on
lymphocyte counts or BMI, a low number of under-
weight patients (total n 5 13), and an observational
period limited to 6 (German) or 24 (Swedish) months
might have affected our results. Nevertheless, despite the
heterogeneous design (prospective study in Germany vs
register-based Swedish cohort) and missing data, sig-
nificant differences or consistent directions were
observed in 2 independent cohorts, substantially sup-
porting the validity of our findings.
Our results may be clinically relevant, suggesting
that underweight females and patients with low base-
line lymphocyte counts (,1.6 3 109/L) possibly
should be monitored more closely. Our observation
may in addition be relevant for patients with child-
hood MS, in whom clinical efficacy and safety of
fingolimod have not yet been established. Further
studies with larger patient numbers and extended
study duration are needed to systematically assess
the clinical relevance of our findings.
AUTHOR CONTRIBUTIONS
Study design: C.W., T.D., B.C.K. Statistical analysis: C.W., R.R. Acqui-
sition and analysis of data: C.W., C.H., N.N., K.W., V.I.L., T.O., B.C.K.
Manuscript draft: C.W., T.D., B.C.K. Critical revision: all authors.
STUDY FUNDING
This work was supported by grants from the German Ministry for Edu-
cation and Research (BMBF, German Competence Network Multiple
Sclerosis [KKNMS], Natalizumab-Pharmakovigilanzstudie, 01GI1002;
to B.C.K.). The research leading to these results has received support
from the Innovative Medicines Initiative Joint Undertaking under grant
agreement 115303, resources of which are composed of financial contri-
bution from the European Union’s Seventh Framework Programme
(FP7/2007–2013) and EFPIA companies’ in kind contribution.
DISCLOSURE
C. Warnke has received speakers honoraria from Bayer HealthCare.
T. Dehmel has received travel expenses from Novartis Pharma and
Genzyme. R. Ramanujam, C. Holmen, N. Nordin, and K. Wolfram
report no disclosures relevant to the manuscript. V. Leussink has
received honoraria for lecturing and financial support for research
from Biogen Idec and Novartis. H. Hartung has received honoraria
for consulting, lecturing, travel expenses for attending meetings, and
financial support for research from Bayer HealthCare, Biogen Idec,
Genzyme, GeNeuro, Merck Serono, Novartis, Roche, Sanofi-
Aventis, and TEVA with approval by the president of Heinrich-
Heine University. T. Olsson has received unrestricted grants and
compensation for participation in advisory boards or lectures from
Novartis, Biogen Idec, and Genzyme. B. Kieseier has received hono-
raria for lecturing, travel expenses for attending meetings, and finan-
cial support for research from Bayer HealthCare, Biogen Idec, Merck
Serono, Novartis, Genzyme, and TEVA. Go to Neurology.org for full
disclosures.
Received April 4, 2014. Accepted in final form September 3, 2014.
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