Sustained Low Relapse Rate With Highly Variable B-Cell Repopulation Dynamics With Extended Rituximab Dosing Intervals in Multiple Sclerosis

Abstract

Background and Objectives
B cell–depleting therapies are highly effective in relapsing-remitting multiple sclerosis (RRMS) but are associated with increased infection risk and blunted humoral vaccination responses. Extension of dosing intervals may mitigate such negative effects, but its consequences on MS disease activity are yet to be ascertained. The objective of this study was to determine clinical and neuroradiologic disease activity, as well as B-cell repopulation dynamics, after implementation of extended rituximab dosing in RRMS.

Methods
We conducted a prospective observational study in a specialized-care, single-center setting, including patients with RRMS participating in the COMBAT-MS and MultipleMS observational drug trials, who had received at least 2 courses of rituximab (median follow-up 4.2 years, range 0.1–8.9 years). Using Cox regression, hazard ratios (HRs) of clinical relapse and/or contrast-enhancing lesions on MRI were calculated in relation to time since last dose of rituximab.

Results
A total of 3,904 dose intervals were accumulated in 718 patients and stratified into 4 intervals: <8, ≥8 to 12, ≥12 to 18, and ≥18 months. We identified 24 relapses of which 20 occurred within 8 months since previous infusion and 4 with intervals over 8 months. HRs for relapse when comparing ≥8 to 12, ≥12 to 18, and ≥18 months with <8 months since last dose were 0.28 (95% CI 0.04–2.10), 0.38 (95% CI 0.05–2.94), and 0.89 (95% CI 0.20–4.04), respectively, and thus nonsignificant. Neuroradiologic outcomes mirrored relapse rates. Dynamics of total B-cell reconstitution varied considerably, but median total B-cell counts reached lower level of normal after 12 months and median memory B-cell counts after 16 months.

Discussion
In this prospective cohort of rituximab-treated patients with RRMS exposed to extended dosing intervals, we could not detect a relation between clinical or neuroradiologic disease activity and time since last infusion. Total B- and memory B-cell repopulation kinetics varied considerably. These findings, relevant for assessing risk-mitigation strategies with anti-CD20 therapies in RRMS, suggest that relapse risk remains low with extended infusion intervals. Further studies are needed to investigate the relation between B-cell repopulation dynamics and adverse event risks associated with B-cell depletion.

Full text

RESEARCH ARTICLE OPEN ACCESS
Sustained Low Relapse Rate With Highly Variable
B-Cell Repopulation Dynamics With Extended
Rituximab Dosing Intervals in Multiple Sclerosis
Chiara Starvaggi Cucuzza, MD, Elisa Longinetti, PhD, Nicolas Ruffin, PhD, Bj¨
orn Evertsson, MD,
Ingrid Kockum, PhD, Maja Jagodic, PhD, Faiez Al Nimer, MD, PhD, Thomas Frisell, PhD,
and Fredrik Piehl, MD, PhD
Neurol Neuroimmunol Neuroinflamm 2023;10:e200056. doi:10.1212/NXI.0000000000200056
Correspondence
Dr. Piehl
fredrik.piehl@ki.se
Abstract
Background and Objectives
B cell–depleting therapies are highly effective in relapsing-remitting multiple sclerosis (RRMS)
but are associated with increased infection risk and blunted humoral vaccination responses.
Extension of dosing intervals may mitigate such negative effects, but its consequences on MS
disease activity are yet to be ascertained. The objectiveof this study was to determine clinical and
neuroradiologic disease activity, as well as B-cell repopulation dynamics, after implementation of
extended rituximab dosing in RRMS.
Methods
We conducted a prospective observational study in a specialized-care, single-center setting,
including patients with RRMS participating in the COMBAT-MS and MultipleMS observational
drug trials, who had received at least 2 courses of rituximab (median follow-up 4.2 years, range
0.1–8.9 years). Using Cox regression, hazard ratios (HRs) of clinical relapse and/or contrast-
enhancing lesions on MRI were calculated in relation to time since last dose of rituximab.
Results
A total of 3,904 dose intervals were accumulated in 718 patients and stratified into 4 intervals:
<8, ≥8 to 12, ≥12 to 18, and ≥18 months. We identified 24 relapses of which 20 occurred within
8 months since previous infusion and 4 with intervals over 8 months. HRs for relapse when
comparing ≥8 to 12, ≥12 to 18, and ≥18 months with <8 months since last dose were 0.28 (95%
CI 0.04–2.10), 0.38 (95% CI 0.05–2.94), and 0.89 (95% CI 0.20–4.04), respectively, and thus
nonsignificant. Neuroradiologic outcomes mirrored relapse rates. Dynamics of total B-cell
reconstitution varied considerably, but median total B-cell counts reached lower level of normal
after 12 months and median memory B-cell counts after 16 months.
Discussion
In this prospective cohort of rituximab-treated patients with RRMS exposed to extended dosing
intervals, we could not detect a relation between clinical or neuroradiologic disease activity and
time since last infusion. Total B- and memory B-cell repopulation kinetics varied considerably.
These findings, relevant for assessing risk-mitigation strategies with anti-CD20 therapies in
RRMS, suggest that relapse risk remains low with extended infusion intervals. Further studies
are needed to investigate the relation between B-cell repopulation dynamics and adverse event
risks associated with B-cell depletion.
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From the Department of Clinical Neuros cience (C.S.C., E.L., N.R., B.E., I.K ., M.J., F.A.N., F.P.), Karolinska Institutet, Stockholm, Sweden; Center for Molecular Medicine (C.S.C., N.R., I.K.,
M.J., F.A.N., F.P.), Karolinska University Hospital, Sto ckholm, Sweden; Department of Neurology (B.E., F.P.), Karolinsk a University Hospital, Stockholm, Sweden; Center for Neurolog y
(C.S.C., I.K., M.J., F.A.N., F.P.), Academic Specialist Center, Stockhol m, Sweden; and Clinical Epidemiology Divis ion (T.F.), Department of Medicine Soln a, Karolinska Institutet,
Stockholm, Sweden.
Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article.
The Article Processing Charge was funded by the Swedish Research Council.
This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1

Multiple sclerosis (MS) is a chronic, inflammatory, de-
myelinating disease of the CNS, affecting 2.8 million people
worldwide.
1
Most patients initially present with acute/subacute
episodes of neurologic deficit, with variable degree of re-
versibility, followed by a period of clinical stability, thus classified
as relapsing-remitting MS (RRMS). Accumulating evidence
demonstrates that B cell–depleting therapies are associated with
strong suppression of RRMS inflammatory disease activity.
2-4
So
far, 2 anti-CD20 monoclonal antibodies, ocrelizumab and ofa-
tumumab, have been approved for use in RRMS in the United
States and European Union. In addition, rituximab, an older
chimeric monoclonal antibody approved for rheumatoid arthritis
and other indications, is increasingly being used off-label in some
countries, including Sweden. The safety profile of anti-CD20
therapies in RRMS comprises an increased infection risk,
5
in-
cluding worsened COVID-19 outcomes.
6-8
Furthermore, anti-
CD20s blunt humoral responses to vaccinations,
9
including for
SARS-CoV-2.
10,11
Collectively, these data warrant studies ex-
ploring risk-mitigation strategies to ensure an optimized benefit-
risk balance for patients with RRMS treated with anti-CD20s. So
far, however, such efforts comprise relatively small-sized studies
with limited dosing interval prolongation and short observation
time.
12,13
Larger real-world cohort studies with sufficiently long
observation time to determine time to normalization of B-cell
levels and possible relation to disease activity and risk of adverse
events are thus lacking. Considering an emerging safety signal
regarding infections already before the COVID-19 pandemic, a
pragmatic anti-CD20 dose extension program was initiated at the
Academic Specialist Center in the fall of 2018 and further ex-
tended with the pandemic outbreak in 2020. The aims of this
study were to determine whether prolonged rituximab dosing
intervals increase the risk of RRMS disease activity and to de-
termine B-cell repopulation dynamics in relation to infusions.
Methods
Study Population
The study population at the Academic Specialist Center
(Stockholm, Sweden) included 718 patients with RRMS enrolled
in either of 2 prospective observational drug trials, COMBAT-
MS (n = 658) and MultipleMS (n = 60), who had been exposed
to at least 2 doses of rituximab by September 1, 2021 (Figure 1A).
The COMBAT-MS study included patients with RRMS initiat-
ing a first disease-modifying therapy (DMT) or doing a
first DMT switch between January 1, 2011, and October 31,
2018. The MultipleMS study included newly diagnosed,
treatment-naive patients with RRMS who initiated a first DMT
between April 1, 2018, and September 28, 2020. Data regarding
demographics, disease, and treatment history were extracted from
the Swedish MS registry. Data on total B-cell count and per-
centage of B cells subpopulations were extracted from medical
records from January 1, 2018, to September 1, 2021.
Standard Protocol Approvals, Registrations,
and Patient Consents
COMBAT-MS, EudraCT: 2016-003587-39, Clinicaltrials.gov
identifier: NCT03193866; MultipleMS, EudraCT: 2017-
002634-24. Written informed consent was obtained from all
study participants (Stockholm Regional Ethics Board, no.
2017/32-31/4 and 2017/1323-31).
Follow-up, Outcomes, and Covariates
Patients were followed annually with assessment of Expanded
Disability Status Scale (EDSS), relapse history, MRI, and acute
contacts when needed. The MRI protocol has been published
previously and considers contrast administration for follow-up
scans as optional in caseof a stable patient,leaving the decision
to the treating neurologist.
14
COMBAT-MS SMSReg data
have been validated against medical records for completeness,
and both studies are continuously monitored for data com-
pleteness.
15
The outcomes in the main analysis were clinical
relapses and brain or spinal cord contrast-enhancing lesions
(CELs). Because annual MRI scans were not synchronized
with rituximab infusions, it was not possible to attribute the
occurrence of new or enlarged T2 lesions to a specific infusion
interval; hence, data are presented for each participant
according to the longest interval ever experienced.
The effect of dose interval extension was assessed by comparing
relapse rates during <8, ≥8to12,≥12 to 18, and ≥18 months
since last infusion. Treatment interval was analyzed as a time-
varying covariate. As a result, treatment intervals longer than 8
months were spilt in more than 1 category, where for example a
14 months interval since last rituximab infusion contributed
data to the <8, ≥8and≥12 months’time bands. The interval
after the first rituximab infusion (or after the first 2 infusions if
<90 days apart) was excluded from the analysis to avoid the
effect of residual disease activity early after treatment start
(eFigure 1, http://links.lww.com/NXI/A770).
The following were considered potential confounders: sex,
age at infusion, EDSS at infusion, number of previous ritux-
imab doses, number of clinical relapses in the year before
rituximab start, number of brain MRI T2 lesions at rituximab
start (categorized as 0, 1–9, 10–20, and >20), and previous
DMTs, classified as none, moderately effective (injectables,
dimethyl fumarate, teriflunomide, or daclizumab), highly ef-
fective (fingolimod, natalizumab, or ocrelizumab), and others
(unspecified).
Glossary
CEL = contrast-enhancing lesion; DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; HR = hazard
ratio; LLN = lower limit of normal; MS = multiple sclerosis; RR = rate ratio; RRMS = relapsing-remitting MS; SPMS =
secondary progressive MS.
2Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN

B-Cell Data
TotalB-cell(CD3
+
CD19
+
) levels were assessed by
flow cytometry before each rituximab infusion, as per
clinical routine, at the Department of Clinical Immunol-
ogy, Karolinska University Hospital. B memory cell
(CD3
+
CD19
+
CD27+immunoglobulin D (IgD)-, CD27+-
IgD+, and CD27−IgD−) percentages were determined in
patients with detectable B cells and converted to absolute
numbers using the extracted data. B-cell data were assessed in
relation to time since last infusion (in months). In instances
where multiple measurements had been performed in the
same treatment interval, only the last assessment was in-
cluded. B-cell samples were classified as depleted if below the
detection level (10 cells/μL for total B and 0.05 cells/μL for
total and CD27+IgD−memory B), partially repleted if above
the detection limit, but below lower limit of normal (LLN, 80
cells/μL for total B, 15.2 cells/μL for total memory B, and 5.6
cells/μL for CD27+IgD−memory B), and completely reple-
ted if equal or above LLN.
Statistical Analysis
Cox proportional hazard regression models were used to
calculate hazard ratios (HRs) and corresponding 95% CIs of
clinical relapse or CELs in relation to rituximab dose intervals.
Time since disease onset was the underlying time scale in all
models; thus, HRs were compared across patients with the
same disease duration. Study entry occurred at each rituximab
infusion, with left truncation of follow-up time to avoid im-
mortal time bias (i.e., we used the counting process approach,
with a start and a stop defining each patient’s each interval).
Every time-to-censoring/outcome after a rituximab dose was
subsequently split in the aforementioned time bands (<8, ≥8
to 12, ≥12 to 18, and ≥18 months), and HRs were calculated
for ≥8 to 12, ≥12 to 18, and ≥18 months since last dose, using
<8 months as reference (for further details, see eMethods,
http://links.lww.com/NXI/A770). A sandwich estimator was
used to account for exposure of the same patient to multiple
rituximab dosing intervals. Models were separately analyzed
as crude and adjusted for confounders (listed above). Cen-
soring events were a subsequent rituximab infusion, conver-
sion to secondary progressive MS (SPMS), emigration, death,
or September 1, 2021, whichever came first. In the main
analysis, we did not include date of switch to a different DMT
as a censoring event in keeping with the hypothesis of a long-
lasting effect of B-cell depletion and to maximize sensitivity
for relapses. However, DMT switch (33 patients out of 718;
4.6%) was considered an additional censoring event in a
sensitivity analysis to restrict the analysis time to exclusive
exposure to rituximab. Negative binomial regression models
were used to calculate repletion rate ratios (RRs) for total B-
and memory B-cell counts per microliter in relation to months
since last infusion. Both total B/memory B-cell counts per
microliter and months since last rituximab infusion were
regarded as numerical, continuous variables in the regression
model. Stratification of the regression model by sex, age, body
mass index (BMI), disease duration, and number of previous
rituximab doses was performed to uncover possible effect
modifiers, with continuous variables transformed in binary
variables taking the approximate median value as splitting
point. With this approach, age was categorized as <40 and ≥40
years, BMI as <24 and ≥24 kg/m
2
, disease duration as <8 and
≥8 years since disease onset, and number of previous ritux-
imab doses as 1–3or≥4. Analyses were conducted with
STATA/BE software, 17.0.
Figure 1 Study Population
Inclusion criteria flowchart. ASC = Academic Specialist Cen-
ter; DMT = disease-modifying therapy, RRMS = relapsing-re-
mitting MS; RTX = rituximab; SMSReg = Swedish MS Registry.
Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 3

Data Availability
Deidentified or aggregated data will be shared with qualified
investigators on reasonable request and pending a relevant data
transfer agreement, in compliance to European legislation.
Results
Study Participants
We identified 718 unique patients with RRMS enrolled in either
COMBAT-MSorMultipleMSstudies who had been exposed to
at least 2 courses of rituximab treatment, for a total of 4622
treatment episodes (Figure 1A and eFigure 2a, http://links.lww.
com/NXI/A770). Patients were followed from each rituximab
infusion until subsequent infusion, occurrence of clinical relapse
or CELs, emigration (n = 1), death (n = 3), transition to SPMS
(n = 19), or end of follow-up, whichever came first. After ex-
clusion of the first treatment course, these individuals had ac-
cumulated 3,904 rituximab dose intervals, for a median follow-up
of 4.2 years (interquartile range 2.7–5.6 years). The baseline
characteristics of study participants are summarized in Table 1.
The infusion-to-outcome/censoring intervals were distrib-
uted as follows: 2,577 intervals (66%) <8 months, 585 (15%)
≥8–12 months, 323 (8%) ≥12–18 months, and 419 (11%)
≥18 months. Importantly, 95% of patients (n = 683) un-
derwent at least 1 interval extension (≥8 months), with 87%
(n = 628) and 56% (n = 403) exposed to an interval of ≥12
months and ≥18 months, respectively. Of the remaining 35
participants, the main reasons for remaining on a <8 months
treatment schedule were (1) censoring event before imple-
mentation of extension protocol, n = 16 (due to conversion to
SPMS, n = 15, or death, n = 1); (2) receiving a second course
of rituximab after January 1, 2021 (n = 10), consequently too
early to have been exposed to dose interval extension; and (3)
unspecified reasons (n = 9), likely involving personal prefer-
ences of the treating neurologist and/or the patient.
Clinical Relapse Occurrence and Relation With
Time Since Last Rituximab Infusion
During follow-up, a total of 24 relapses were recorded (eFigure 2b,
http://links.lww.com/NXI/A770) with the annualized relapse
rate dropping from a mean of 0.49 (95% CI 0.44–0.54) in the year
Table 1 Cohort Demographic and Clinical Characteristics
Overall (n = 718)
No. of females (%) 511 (71)
Age at onset, median (Q1–Q3) 29 (24–36)
Age at diagnosis, median (Q1–Q3) 32 (26–39)
Age at RTX start, median (Q1–Q3) 37 (30–45)
Disease duration at RTX start (y), median (Q1–Q3) 5.5 (1.9–10.8)
ARR in the year before RTX start, mean (SD) 0.49 (0.67)
EDSS at RTX start, median (Q1–Q3) 2(1–2.5)
No. of brain T2 lesions at RTX start, no. of patients (%)
0 lesions 7(1)
1–9 lesions 141 (20)
10–20 lesions 196 (28)
>20 lesions 359 (51)
Class of previous DMT, no. of patients (%)
None 258 (36)
Moderately effective
a
273 (38)
Highly effective
b
151 (21)
Others
c
36 (5)
No. of RTX doses, median (Q1–Q3) 6(4–8)
Duration of follow-up (y), median (Q1–Q3) 4.2 (2.7–5.6)
Abbreviations: ARR = annualized relapse rate; DMT = disease-modifying therapy; EDSS = Expanded Disability Status Scale; Q1 = first quartile; Q3 = third
quartile; RTX = rituximab.
a
Injectables, teriflunomide, dimethyl fumarate, and daclizumab.
b
Fingolimod, natalizumab, and ocrelizumab
c
Unspecified or study drug.
4Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN

before rituximab start to 0.03 (95% CI 0.02–0.05) in the first
treatment year and ≤0.01 onward, up to 10 years of follow-up
(eFigure 2c, http://links.lww.com/NXI/A770).
We subsequently determined whether longer time since last
rituximab infusion was associated with increased risk of a
clinical relapse. HRs compared with <8 months since last
infusion were 0.28 (95% CI 0.04–2.10), 0.38 (95% CI
0.05–2.94), and 0.89 (95% CI 0.20–4.04) for ≥8 to 12, ≥12 to
18, and ≥18 months, respectively (Table 2). Adjustment for
potential confounders did not substantially divert from the
crude model. Adjusted HRs for ≥8 to 12, ≥12 to 18, and ≥18
months compared with <8 months since last infusion were
0.30 (95% CI 0.04–2.32), 0.42 (95% CI 0.05–3.23), and 0.85
(95% CI 0.18–3.92), respectively.
When considering date of switch to a different DMT as ad-
junctive censoring parameter, 3 relapses were censored,
leaving a total of 21 clinical relapses. In this setting, adjusted
HRs compared with <8 months resulted in 0.32 (95% CI
0.04–2.38) for ≥8–12 months and 0.68 (95% CI 0.07–6.6) for
>18 months since last rituximab dose, whereas HR for
≥12–18 months could not be calculated due to the absence
of events (Table 2). Overall, these point estimates suggest a
similar or lower risk of relapse with transition from a regular to
an extended dosing interval regimen.
Inclusion of Contrast-Enhancing Lesions
as Outcome
Neuroradiologic assessments conducted with administration of
contrast agent (1,370 brain or spinal cord MRI scans out of 3,075
total scans, 44.6%, resulting in 548 patients with at least 1 follow-
up scan with contrast administration) were evaluated. Among
these, 11 scans revealed CEL events, 4 of which were performed
in the same treatment interval of a registered clinical relapse.
When considering both relapse and/or CELs, 31 disease activity
events were recorded, with HRs of 0.24 (95% CI 0.03–1.77),
0.66 (95% CI 0.15–2.93), and 0.72 (95% CI 0.16–3.35) for ≥8to
12, ≥12 to 18, and ≥18 months, respectively, compared with <8
months since last infusion in the crude model. Similar to the
previous analysis considering only clinical relapses, adjustment
for potential confounders barely altered the associations. HRs
were 0.26 (95% CI 0.03–1.96), 0.72 (95% CI 0.17–3.08), and
0.68 (95% CI 0.14–3.24) for ≥8 to 12, ≥12 to 18, and ≥18
months since last dose, respectively (Table 2).
Overall, the risk of an adverse efficacy outcome did not no-
ticeably change up to 3 years after rituximab infusion, with an
Table 2 Hazard Ratios for (1) Relapse, (2) Relapse and/or Contrast-Enhancing Lesions, and (3) Relapse With Disease-
Modifying Treatment Switch as Adjunctive Censoring Parameter, With Extended Time Since Rituximab Infusion
Compared With <8 Months
No. events Person-years Incidence rate
Cox model HR (95% CI)
a
Crude Adjusted
b
Clinical relapses
<8 months 20 2,119.6 0.009
≥8–12 months 1 326.4 0.003 0.28 (0.04–2.10) 0.30 (0.04–2.32)
≥12–18 months 1 262.3 0.003 0.38 (0.05–2.94) 0.42 (0.05–3.23)
≥18 months 2 256.3 0.008 0.89 (0.20–4.04) 0.85 (0.18–3.92)
Clinical relapses and/or CELs
<8 months 26 2,117.4 0.012 . .
≥8–12 months 1 325.8 0.003 0.24 (0.03–1.77) 0.26 (0.03–1.96)
≥12–18 months 2 261.3 0.008 0.66 (0.15–2.93) 0.72 (0.17–3.08)
≥18 months 2 249.7 0.008 0.72 (0.16–3.35) 0.68 (0.14–3.24)
Clinical relapses DMT switch included in censoring
<8 months 19 2,105.3 0.009 . .
≥8–12 months 1 319.9 0.003 0.30 (0.04–2.26) 0.32 (0.04–2.38)
≥12–18 months 0 250.7 0 n/a n/a
≥18 months 1 207.6 0.005 0.65 (0.08–5.21) 0.68 (0.07–6.60)
Abbreviations: CEL = contrast-enhancing lesion; HR = hazard ratio.
a
Time since disease onset as underlying time scale of all models.
b
Adjusted for sex, age at infusion, Expanded Disability Status Scale at infusion, previous disease-modifying therapies, number of brain MRI T2 lesions at
rituximab start, annualized relapse rate in the year before rituximab start, and number of previous rituximab doses.
Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 5

event-free survival rate of 0.98 (95% CI 0.97–0.99)
(Figure 2A). In the same manner, the incidence rate for re-
lapse and/or CELs remained stable across the 4 time bands
(Table 2 and Figure 2B).
New or Enlarging T2 Lesions on MRI Scans
Forty-eight MRI scans in 44 patients (6.9% of 636 patients
with valid follow-up and reference MRI scans) displayed new
or enlarging T2 lesions. As yearly scans were not synchronized
with rituximab infusions, the time period covered between
follow-up and reference MRI scans did not match a specific
treatment interval, so data are reported per individual instead
of per dosing interval, with stratification for the longest
treatment interval ever experienced when increase in T2 le-
sion burden was recorded. With this approach, 25 patients
were in the <8 months group (out of 90 patients in this group,
27.8%), whereas 7 (out of 144, 4.8%), 4 (out of 242, 1.7%),
and 8 (out of 160, 5%) were in the ≥8 to 12, ≥12 to 18, and
≥18 months groups, respectively.
Total B-Cell Repopulation Kinetics
B-cell data were available only after January 1, 2018, resulting
in 1,744 data points for a total of 2,208 infusions after this
date in 648 patients (79% of the infusions; 90% of the patient
cohort). The median of total B-cell counts reached detect-
able levels 6 months since last infusion and the LLN after 12
months (Figure 3A). However, a considerable degree of
variability of total B-cell levels was observed, with variance
increasing over time since last infusion and a small pro-
portion of subjects (3.4%) remaining completely depleted
even with the longest dosing intervals. When considering the
same time bands used in the main analysis, we found that
63.9% of samples remained depleted in the <8 months
group. In contrast, the majority of samples were partially or
completely repleted in the extended-interval groups. The 2
categories combined accounted for 87.1%, 97.9%, and 96.6%
of the samples in the ≥8 to 12, ≥12 to 18, and ≥18 months
groups, respectively (Figure 3B). Negative binomial re-
gression confirmed positive association between B-cell levels
and months since last infusion, with a repletion RR of 1.17
(95% CI 1.14–1.19, p< 0.0001). In other words, the model
predicted an increase in B-cell counts of 17% every month in
the analyzed data set. When stratifying according to age, sex,
BMI, disease duration, or number of previous rituximab
doses, we did not observe any significant effect modification
by sex, age, and BMI, whereas longer disease duration and
higher number of previous rituximab doses were associated
with a slower rate of B cell repopulation, as expressed by the
RR (interaction coefficients 0.94, 95% CI 0.9–0.98 and 0.9,
95% CI 0.86–0.95 for disease duration and number of pre-
vious doses, respectively). The stratified RRs were 1.21 (95%
CI 1.18–1.25) and 1.14 (95% CI 1.12–1.16) for disease
duration <8 or ≥8 years, respectively, and 1.24 (95% CI
1.19–1.30) and 1.12 (95% CI 1.10–1.15) for B-cell repo-
pulating after infusions preceded by 1–3or≥4 rituximab
doses, respectively (Figure 3, C and D).
Memory B-Cell Repopulation Kinetics
Memory B-cell data were available only for samples with de-
tectable B cells. In this subgroup of 1,157 samples, 580 sam-
ples (46%) also included B-cell subpopulation analysis, for a
total of 489 patients. Compared with total B-cell levels,
memory B cells showed a slower repopulation kinetic: the
median of memory B-cell counts, categorized in 1-month time
bands, was steadily above the limit of detection but reached
LLN only after 16 months (Figure 4A). Thus, memory B cells
were partially repleted in the majority of samples in the first 2
time bands (79.7% and 59.5% for the <8 and ≥8–12 months,
Figure 2 Risk of Clinical Relapse and/or Contrast-Enhancing Lesion Occurrence in Relation to Time Since Last Rituximab
Infusion
(A) Kaplan-Meier curve of event-free time
since last rituximab infusion. (B)Incidence rate
of clinical relapse and/or CELs at <8 months,
≥8 to 12, ≥12 to 18, and ≥18 months since last
rituximab infusion. CEL = contrast-enhancing
lesion; RTX = rituximab.
6Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN

respectively), whereas most samples (60.8%) were normal-
ized after 18 months since last infusion (Figure 4B). Ac-
cordingly, when applying a negative binomial regression
model, the repletion rate ratio was 1.03 (95% CI 1.02–1.04,
Figure 4C). No effect modification was observed stratifying
samples according to age, sex, BMI, disease duration, or
number of previous rituximab doses (Figure 4D). When
considering separately the 3 subsets composing memory
B cells (CD27+IgD−, CD27+IgD+ or double positive, DP,
and CD27−IgD−or double negative, DN), we noted dif-
ferent repopulation kinetics, with LLN reached in 12 and 7
months for DP and DN subsets, respectively (Figure 5, A
and B), whereas median levels of CD27+IgD−memory
subpopulation persistently remained below LLN for up to
24 months (Figure 5C). As a result, this subset was only
partially repleted in the majority of samples up to 24 months
since last rituximab infusion: 87.5%, 84%, 77%, and 66.9%
partially repleted samples for <8, ≥8 to 12, ≥12 to 18, and
≥18 months, respectively (Figure 5D).
Discussion
We here related inflammatory disease activity to time since last
rituximab infusion in a large cohort of patients with RRMS
exposed to dose interval extension with long prospective follow-
up. Alike the ocrelizumab label, the Swedish MS Society rec-
ommends rituximab dosing (500 mg) every 6 months. Because
of an emerging signal for increased infection rate over time,
5
from October 2018, the Academic Specialist Center changed
guidelines to recommend dose extension to 12 months after the
fifth infusion for patients with stable disease. With the start of
the COVID-19 pandemic in March 2020, infusion intervals
were further extended to 12–24 months or more from the
second infusion regardless of treatment duration, a decision also
influenced by accumulating evidence suggesting an adverse ef-
fect on COVID-19 outcomes.
6,7
We previously reported the absence of rebound disease ac-
tivity in patients with RRMS who, for various reasons, had
Figure 3 Total B-Cell Repopulation Dynamics in Relation to Time Since Last Rituximab Infusion
(A) Box plot depicting distributions of B-cell count grouping samples into 1-month-time intervals since last rituximab infusion. Continuous red line: B-cell
detection limit; dashed black line: LLN. (B) Total number of observations (y axis) with relative frequencies (bar labels) of depleted (<10 cells/μL), partially
repleted (≥10–80 cells/μL), and completely repleted (≥80 cells/μL) B-cell counts at <8 months, ≥8 to 12, ≥12 to 18, and ≥18 months since last rituximab infusion.
(C) Scatter plot of total B-cell counts in relation to time since last infusion, with samples collected within 1 month from a relapse/contrast-enhancing lesion
highlighted in red. The predicted repopulation kinetic up to 18 months since last rituximab infusion according to a negative binomial regression model is
depicted by the black curve. Continuous red line: B-cell detection limit; dashed black line: lower limit of normal. (D) Coefficient plot of the repletion rate ratios
(RRs) for B-cell repopulation for the overall model and stratified according to sex, age (<40 and ≥40 years of age), BMI (<24 and ≥24 kg/m
2
), disease duration (<8
and ≥8 years), and number of previous rituximab doses (1–3 and ≥4). Among the analyzed potential effect modifiers, disease duration and number of previous
rituximab doses affected B-cell reappearance kinetics (Wald test p< 0.01 for disease duration, **, and p< 0.000 1 for rituximab doses, ****). #BMI data were
available for 86.7% of the patients, 86.5% of the samples.
Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 7

stopped rituximab for more than 12 months.
16
Similar results
were reported for patients with RRMS exposed to low dose
(<1000 mg yearly) or stopping treatment in another Swedish
study.
17
Furthermore, based on data from the phase II trial of
ocrelizumab, which included a safety follow-up of about 12
months without treatment, it has been suggested that treat-
ment intervals could be extended well beyond the regular 6-
month interval.
18
More recently, a retrospective observational
study did not detect signs of disease activity rebound with
extension of ocrelizumab intervals by 4 weeks or more.
12
Fi-
nally, a Dutch prospective study implemented a personalized
dosing interval of ocrelizumab based on B cell counts.
13
In this
study B-cell levels were assessed monthly after 6 months from
the last infusion with retreatment with counts ≥10 cells/μL,
which occurred at a median of 34 weeks since last infusion.
However, all these studies were relatively small and had
considerably shorter follow-up periods than included here,
with a median structured prospective follow-up of 4.2 years
(interquartile range 2.7–5.6 years).
Of interest, we could not detect an increased risk of
clinical relapses and/or CELs for any of time bands ana-
lyzed, i.e., ≥8 to 12, ≥12 to 18, and ≥18 months, with point
estimates of relapse risk remaining remarkably stable.
Although the power to detect a difference was limited
due to the limited number of relapse/CEL events, our
observations suggest that both relapse risk and neuro-
radiologic disease activity remain low well beyond regular
infusion intervals. The fact that HRs with extended dosing
intervals in all cases were below 1 compared with the
regular interval is reassuring, but likely can be influenced by
overall treatment duration and a small number of subjects
switching treatment or being held on shorter infusion intervals.
It is important to note, however, that a substantial enrichment
of stable patients in the longer time bands is unlikely given the
prospective study design and the high proportion of partici-
pants being exposed to extended intervals. Taken together,
these results are valuable for designing further, sufficiently
powered confirmatory studies, but meanwhile provide
Figure 4 Memory B-Cell Repopulation Dynamics in Relation to Time Since Last Rituximab Infusion
(A) Box plot showing distributions of memory B-cell count grouping samples into 1-month time intervals since last rituximab infusion. Continuous red line:
memory B-cell detection limit; dashed black line: lower limit of normal. (B) Total number of observations (y axis) with relative frequencies (bar labels) of
depleted (<0.05 cells/μL), partially repleted (≥0.05–15.2 cells/μL), and completely repleted (≥15.2 cells/μL) levels of memory B cells at <8 months, ≥8 to 12, ≥12
to 18, and ≥18 months since last rituximab infusion. (C) Scatter plot of memory B-cell counts in relation to time since last infusion, with samples collected
within 1 month from a relapse/contrast-enhancing lesion highlighted in red. The repopulation kinetic predicted by a negative binomial regression model up to
24 months since last rituximab infusion is shown by the black curve. Continuous red line: memory B-cell detection limit; dashed black line: lower limitof
normal. (D) Coefficient plot of the repletion rate ratios (RRs) for memory B-cell repopulation for the overall model and stratified according to sex, age, BMI,
disease duration, and number of previous rituximab doses. None of the models showed any effect of the analyzed covariates on memory B-cell repletion
rates.
#
BMI data were available for 88.1% of the patients, 86.9% of the samples. BMI = body mass index
8Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN

preliminary results indicating a viable approach for improving
the benefit-risk balance with B cell–depleting DMTs. This is
especially relevant in context of increased susceptibility to in-
fections, lowered immunoglobulin levels, scheduling of vacci-
nations, or planning of pregnancy.
5,10,19,20
The striking efficacy of anti-CD20 therapies underscores the
role of B cells in MS disease pathogenesis. Experiences across
a wide spectrum of autoimmune conditions indicate numer-
ous ways by which B cells can contribute to disease, which also
depends on the type of disease process.
21
In MS, B cells have
been shown to produce cytokines with a presumed role in
supporting CNS inflammation, with supporting functional
data obtained in experimental autoimmune encephalitis, an
animal model of MS.
22,23
Notably, however, antigen-specific
memory B-cell clones have also been shown to activate
memory T cells with encephalitogenic features in subjects
with RRMS ex vivo.
24
The possible implication of this ob-
servation is that disease-driving cells mainly belong to the
memory B-cell subtype, alike what has been suggested for
neuromyelitis optica, where monitoring of memory B-cell
subset has been suggested to be useful to determine anti-
CD20 infusion intervals.
25,26
However, it is important to
acknowledge that clinical effectiveness of extended dosing
intervals cannot readily be extrapolated due to different
pathomechanisms, whereas it is more likely that B-cell repo-
pulation kinetics will be more similar across the 2 conditions.
However, in spite of increasing use of anti-CD20 therapies in
MS, there is only limited information on the kinetics of B-cell
repopulation after depletion and if there is any relation with
return of disease activity. Insights regarding different memory
B-cell subsets are also lacking. To this end, we analyzed total
B-cell and memory B-cell counts determined in clinical rou-
tine for our cohort. Although limited by a certain irregularity
of assessments in this real-world setting, our data, neverthe-
less, demonstrate a considerable variability in reconstitution
kinetics of total B-cell numbers, where median counts were
normalized after 12 months, whereas memory B cells
remained below LLN for up 16 months since last dose. In the
prediction model we used, based on negative binomial re-
gression, total B-cell counts increased by 17% every month,
with disease duration and number of previous rituximab
doses negatively affecting repopulation rates, although re-
sults should be interpreted with some caution due to the
variability in timing of sampling. Regarding memory B cells,
the repletion rate was much slower, with a monthly increase
Figure 5 Memory B Subpopulation Repopulation Dynamics
(A–C) Box plot showing distributions of the 3 subsets of memory B cell: CD27+IgD−(A), CD27+IgD+ or double positive (DP, B), and CD27−IgD−or double
negative (DN, C). Samples were grouped into 1-month time intervals since last rituximab infusion. Continuous red line: memory B-cell detection limit (0.05
cells/μL for all subsets); dashed black line: lower limit of normal (5.6 cells/μL for CD27+IgD−cells, 3.44 cells/μL for DP cells and 1.92 cells/μL for DN cells). (D)
Total number of observations (y axis) with relative frequencies (bar labels) of depleted (<0.05 cells/μL), partially repleted (≥0.05–5.6 cells/μL), and completely
repleted (≥5.6 cells/μL) levels of CD27+IgD−memory B cells at <8 months, ≥8 to 12, ≥12 to 18, and ≥18 months since last rituximab infusion.
Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 9

of only 3%. When considering different memory B-cell
subsets, CD27−IgD−were the first to repopulate, followed
by CD27+IgD+, whereas median levels of CD27+IgD−
remained consistently below LLN up to 24 months since last
infusion. Overall, these results might hint at different roles of
memory B-cell subsets in RRMS pathogenesis. The low
number of adverse efficacy events coupled with lack of sys-
tematic determination of B cells at occurrence of clinical or
radiologic activity meant that a comparison between non-
active and active patients could not be performed. However,
it is evident that a strong signal for return of inflammatory
disease activity with repletion of B cells, including of the
memory subtype, is lacking.
Apart from previously mentioned limitations relating to the
real-world nature of this study, including incomplete data
coverage and variability in the structure of data collection, this
study also did not include volumetric MRI data or soluble
biomarkers such as neurofilament light chain concentrations.
Thus, we cannot exclude that dose interval extension nega-
tively affects disease processes not reflected by relapses or
accrual of focal MRI lesions, and studies exploring the effect of
extended B cell–depleting treatment schedules on the pro-
gressive aspects of MS are therefore warranted.
In summary, our findings suggest that anti-CD20 dose in-
terval extension could be considered in patients with RRMS
with stable disease without incurring risk of return of in-
flammatory disease activity in the short to medium term,
especially in case of treatment-related adverse events or
when planning pregnancy. Further studies are needed to
determine whether dose interval extension is also associated
with a lowered risk of infection, while it has been shown that
vaccination responses are improved with B-cell repopula-
tion,
27
in turn improving benefit-risk with anti-CD20
therapies.
Acknowledgment
The authors thank all the patients participating in the
COMBAT-MS and the MultipleMS studies and Simon
Englund, BSc, and Tommaso Piehl for their help with data
collection.
Study Funding
This work was funded by the Patient-Centered Outcomes
Research Institute (PCORI) Award (Combat-MS, MS-
1511-33196), Swedish MRC grant no. 2020-02700, the
European Union’s Horizon 2020 Research and Innovation
Programme (MultipleMS, EU RIA 733161), the Knut and
Alice Wallenberg Foundation, the Swedish Research
Council for Health, Working Life, and Welfare (postdoc
Grant No: 2020-0115 to E.L.), and the Swedish Brain
Foundation.
Disclosure
F. Piehl has received research grants from Merck KGaA and
UCB and fees for serving on DMC in clinical trials with
Chugai, Lundbeck, and Roche. C. Starvaggi Cucuzza has
received a travel grant from SanofiGenzyme. B. Evertsson
has received travel support from Roche. The remaining
authors declare no competing interests. Go to Neurology.
org/NN for full disclosures.
Publication History
Received by Neurology: Neuroimmunology & Neuroinflammation
May 10, 2022. Accepted in final form September 16, 2022. Submitted
and externally peer reviewed. The handling editor was Friedemann Paul,
MD.
Appendix Authors
Name Location Contribution
Chiara
Starvaggi
Cucuzza, MD
Department of Clinical
Neuroscience, Karolinska
Institutet, Stockholm, Sweden;
Center for Molecular Medicine,
Karolinska University Hospital,
Stockholm, Sweden
Drafting/revision of the
manuscript for content,
including medical writing
for content; major role in
the acquisition of data;
study concept or design;
and analysis or
interpretation of data
Elisa
Longinetti,
PhD
Department of Clinical
Neuroscience, Karolinska
Institutet, Stockholm, Sweden
Drafting/revision of the
manuscript for content,
including medical writing
for content; study concept
or design; and analysis or
interpretation of data
Nicolas
Ruffin, PhD
Center for Molecular Medicine,
Karolinska University Hospital,
Stockholm, Sweden; Center for
Molecular Medicine, Karolinska
University Hospital, Stockholm,
Sweden
Drafting/revision of the
manuscript for content,
including medical writing
for content; study concept
or design; and analysis or
interpretation of data
Bj¨
orn
Evertsson,
MD
Department of Clinical
Neuroscience, Karolinska
Institutet, Stockholm, Sweden;
Department of Neurology,
Karolinska University Hospital,
Stockholm, Sweden
Drafting/revision of the
manuscript for content,
including medical writing
for content, and major
role in the acquisition of
data
Ingrid
Kockum,
PhD
Department of Clinical
Neuroscience, Karolinska
Institutet, Stockholm, Sweden;
Center for Molecular Medicine,
Karolinska University Hospital,
Stockholm, Sweden; Center for
Neurology, Academic Specialist
Center, Stockholm, Sweden;
Drafting/revision of the
manuscript for content,
including medical writing
for content, and major
role in the acquisition of
data
Maja
Jagodic, PhD
Department of Clinical
Neuroscience, Karolinska
Institutet, Stockholm, Sweden;
Center for Molecular Medicine,
Karolinska University Hospital,
Stockholm, Sweden; Center for
Neurology, Academic Specialist
Center, Stockholm, Sweden;
Drafting/revision of the
manuscript for content,
including medical writing
for content, and major
role in the acquisition of
data
Faiez Al
Nimer, MD,
PhD
Department of Clinical
Neuroscience, Karolinska
Institutet, Stockholm, Sweden;
Center for Molecular Medicine,
Karolinska University Hospital,
Stockholm, Sweden; Center for
Neurology, Academic Specialist
Center, Stockholm, Sweden;
Drafting/revision of the
manuscript for content,
including medical writing
for content; major role in
the acquisition of data;
and study concept or
design
10 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 Neurology.org/NN

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Appendix (continued)
Name Location Contribution
Thomas
Frisell, PhD
Clinical Epidemiology Division,
Department of Medicine Solna,
Karolinska Institutet,
Stockholm, Sweden
Drafting/revision of the
manuscript for content,
including medical writing
for content; study concept
or design; and analysis or
interpretation of data
Fredrik
Piehl, MD,
PhD
Department of Clinical
Neuroscience, Karolinska
Institutet, Stockholm, Sweden;
Center for Molecular Medicine,
Karolinska University Hospital,
Stockholm, Sweden;
Department of Neurology,
Karolinska University Hospital,
Stockholm, Sweden; Center for
Neurology, Academic Specialist
Center, Stockholm, Sweden;
Drafting/revision of the
manuscript for content,
including medical writing
for content; major role in
the acquisition of data;
study concept or design;
and analysis or
interpretation of data
Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 1 | January 2023 11

DOI 10.1212/NXI.0000000000200056
2023;10; Neurol Neuroimmunol Neuroinflamm
Chiara Starvaggi Cucuzza, Elisa Longinetti, Nicolas Ruffin, et al.
With Extended Rituximab Dosing Intervals in Multiple Sclerosis
Sustained Low Relapse Rate With Highly Variable B-Cell Repopulation Dynamics
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