Accepted Manuscript

Evaluation of Responsiveness to Reduced-dose Rituximab in

Corticotropin/IVIg/Rituximab Combination Immunotherapy for
Opsoclonus-Myoclonus Syndrome

Michael R. Pranzatelli , Elizabeth D. Tate , Nathan R. McGee ,

Craig A. MacArthur

PII: S0887-8994(18)30341-2
DOI: 10.1016/j.pediatrneurol.2018.05.003
Reference: PNU 9350

To appear in: Pediatric Neurology

Received date: 10 April 2018

Accepted date: 6 May 2018

Please cite this article as: Michael R. Pranzatelli , Elizabeth D. Tate , Nathan R. McGee ,
Craig A. MacArthur , Evaluation of Responsiveness to Reduced-dose Rituximab in Corti-
cotropin/IVIg/Rituximab Combination Immunotherapy for Opsoclonus-Myoclonus Syndrome, Pediatric
Neurology (2018), doi: 10.1016/j.pediatrneurol.2018.05.003

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 ACCEPTED MANUSCRIPT

Pediatric Neurology

Clinical Observations

Evaluation of Responsiveness to Reduced-dose

Rituximab in Corticotropin/IVIg/Rituximab

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Combination Immunotherapy for Opsoclonus-

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Myoclonus Syndrome

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Michael R. Pranzatelli MD a, Elizabeth D. Tate MN, ARNP a, Nathan R. McGee BS a,
Craig A. MacArthur MD b
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Inc., Orlando, Florida

b
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National Pediatric Myoclonus Center and National Pediatric Neuroinflammation Organization,

Department of Pediatric Hematology/Oncology, Golisano Children’s Hospital of Southwest

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Florida, Lee Memorial Health Care System, Fort Myers, Florida
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* Communications should be addressed to: Prof. Dr. M. R. Pranzatelli; National Pediatric

Neuroinflammation Organization, Inc.; 12001 Research Parkway; Suite 236; Orlando, FL 32826
E-mail address: mpranzatelli@omsusa.org
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Abstract word count – 242/250

Text word count – 2043/2000
Tables – 1
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Figures – 1
References – 17/15
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Short running title: FLAIR-CI Therapy for OMS

Keywords: anti-CD20, B cell depletion, CSF B cells, neuroblastoma, opsoclonus-myoclonus

syndrome, paraneoplastic syndrome, pediatric neuroinflammatory disorders, neuroinflammation
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E-mail addresses of co-authors:

E. Tate: etate@omsusa.org
N. McGee: mcgee6510@att.net
C. MacArthur: craig.macarthur@leehealth.org
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ABSTRACT

BACKGROUND: Rituximab (anti-CD20) has been used as B cell-targeted intervention to treat

opsoclonus-myoclonus syndrome (OMS). Due to isolated reports of chronic
hypogammaglobulinemia and B lymphopenia following rituximab in several disorders, and rapid
B cell depletion after a few doses, we reduced the dosage 20% in our clinical practice.
METHODS: In this IRB-approved retrospective study, 32 children with OMS and cerebrospinal
fluid (CSF) B cell expansion had received front-loaded adrenocorticotropic hormone (ACTH1-
39), intravenous immunoglobulin (IVIg), and rituximab combination immunotherapy (―FLAIR-

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CI‖) for de novo OMS. Parametric statistical analysis compared 10 children receiving 1200
mg/m2 of rituximab (300 mg/m2 x 4) and 22 receiving 1500 mg/m2 (375 mg/m2 x 4). Clinical

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response had been video-documented and scored by a blinded observer. RESULTS: In both
groups, motor severity (total score) lessened by ≥ 76% and CSF B cells were similarly depleted

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(≥ 95%) 6 months after treatment. None of the treated patients remained unable to walk
independently. Serum IgM depletion was analogous in the 1200 mg/m2 (-73%) and 1500 mg/m2
group (-64%). The relapse frequency was similar in both groups. Side effects were principally
steroidal, tolerable, and transient. Circulating B cell repopulation was comparable.

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CONCLUSIONS: The reduced-dose of rituximab in FLAIR-CI was as effective and well-
tolerated as the standard dose, and provided rapid, early therapeutic intervention in OMS.
Pending a long-term prospective study, these are proof-of-concept data in support of challenging
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the dose of rituximab in various disorders, which may have different dose requirements.
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Introduction

Rituximab, a first-generation murine-human chimeric monoclonal antibody (anti-CD20), has

been used to treat a wide variety of autoimmune and lymphoproliferative diseases in children.1
One such example is opsoclonus-myoclonus syndrome (OMS). Following the discovery that
cerebrospinal fluid (CSF) B cells and humoral markers are elevated in OMS,2 the use of
rituximab to treat the B cell expansion was shown to be effective immunologically and clinically
in a prospective, phase I, open-label, clinical trial (NCT00244361),3,4 as found in an
observational study (adjunctive)5 and small case series.6-8 Anti-CD20, the first cell-specific

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treatment effective in OMS, works best in combination therapy.3
FLAIR therapy is an acronym coined by The National Pediatric Myoclonus Center for front-

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loaded adrenocorticotropic hormone (ACTH1-39), intravenous immunoglobulin (IVIg), and
rituximab combination immunotherapy (―FLAIR-CI‖).9 The strategy was to use multiple

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immunological agents, presumed to have distinct mechanisms of action, early in the course of
OMS, when immunobiomarkers of disease activity are highest, to enhance the rate of
neurological remission. The clinical imperative warranting intensified immunotherapy in OMS is
the need to induce neurological remission rapidly to decrease neurological and neuropsychiatric

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morbidity, such as cognitive impairment.9 Increased immunosuppression is associated with better
neurodevelopmental outcome in OMS.10,11 Also, OMS relapses in more than half of patients
treated just with steroids or IVIg.12
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The ―standard‖ mode of delivering rituximab is 375 mg/m2 IV weekly for 4 consecutive
weeks (1500 mg/m2).13 However, both the dose and dosing schedule were chosen arbitrarily, i.e,
the supply of rituximab divided by the number of projects. We decided to revisit rituximab dose
because prolonged hypogammaglobulinemia and B lymphopenia can occur in rituximab-treated
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individuals,14,15 as in a few children we had seen, and patient susceptibility cannot be predicted.
Therefore, we reduced the dose by 20% to 300 mg/m2 per infusion (1200 mg/m2) as a precaution
in our practice, knowing that B cell killing is rapid. We report our retrospective clinical and
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immunologic observations on the previously unstudied reduced dose compared to the standard
dose3 as to reduction in OMS severity, rectification of CSF B cell expansion, and OMS relapse.
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Methods

Study design
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Inclusion criteria included untreated patients of either gender with confirmed OMS whether or
not a body cavity neuroblastoma had been found (as in 50% of OMS), and regardless of OMS
severity/duration. Those with questionable diagnosis, prior chemotherapy or immunotherapy, or
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contraindications for lumbar puncture were excluded. Treatment response was defined as
statistically significant decrease in OMS severity as measured by the Opsoclonus-Myoclonus
Evaluation Scale (OMES), and reduction in neuroinflammation as measured by the frequency of
CSF and blood CD19+ B cells. Relapse, defined as re-occurrence of at least one major OMS
symptom or sign for  72 hours, was considered mild if it involved only ataxia, moderate if
opsoclonus also was present, or severe if the child became non-ambulatory.

Patients

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Patients had been evaluated at the National Pediatric Myoclonus Center from 2006 to 2010
and parents signed informed consent for an IRB-approved observational study of immunological
abnormalities in OMS (SIU School of Medicine, Springfield, IL). Blood and CSF were obtained
at initial diagnostic evaluation, utilizing lymphocyte subset analysis for its higher diagnostic
yield than routine CSF studies.2 This was not a clinical trial; treatment was based on clinical
decisions, and drugs were administered by treating physicians in clinical care. Patients with
neuroinflammation were re-consented at approximately 6 months for repeat testing, given the
high neurological and neuropsychiatric morbidity of OMS, to demonstrate no evidence of

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disease activity (NEDA). Western IRB (Puyallup, WA) designated exempt review status for
retrospective analysis of all the data. Table 1 shows that the two groups were very well balanced

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as to demographics and clinical traits.

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Treatments

Patients had received the following biologicals and drugs: 1) rituximab intravenously (IV) at
375 mg/m2 or 300 mg/m2 once weekly for 4 consecutive weeks; 2) ACTH1-39 (H.P. Acthar Gel®

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80 IU/ml) at 75 IU/m2 IM twice a day for one week, daily for one week, on alternate days for
two weeks, then tapered over subsequent months; 3) IVIg (any IgA-deficient brand) was infused
at 1 g/kg on each of two consecutive days for induction, then 1 g/kg once monthly for
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maintenance. Agents were administered consecutively, spaced apart by days or weeks. ACTH
was the first agent to be started in 40% of the 1200 mg/m2 group and 50% of the 1500 mg/m2
group. Rituximab was the third agent delivered in 70% of the 1200 mg/m2 group and 59% of the
1500 mg/m2 group. Infusion pretreatments included acetaminophen and diphenhydramine.
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Prophylactic treatments were trimethoprim/sulfmethoxazole, ranitidine HCl, low sodium diet,

and daily calcium with vitamin D.
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Clinical procedures

By written parental consent, patients had been videotaped before and after treatment using
standardized written criteria for video-documentation of clinical response. History and physical
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examination was performed. Lumbar puncture was done atraumatically as previously described.4
Blood for simultaneous comparisons was drawn by vein or IV port usually 4 weeks, no sooner
than 3 weeks, after the last IVIg infusion.
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Laboratory procedures

Lymphocyte subsets were identified ex vivo by 5-color single laser flow cytometry using
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conjugated monoclonal antibodies to CD3, CD19, and CD45 as previously described. Assays
were performed in the hospital Flow Cytometry Department (St. John’s Hospital, Springfield,
IL) utilizing a Becton-Coulter Cytomics FC500 cytometer. Blood B cell percentage and counts
were referenced to published pediatric norms. Serum Ig was quantitated by Tina-quant assay, an
antigen turbidity test, in the clinical laboratory.

Statistical analysis

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GraphPad software (San Diego, CA) was used for statistics and graphics. The statistical
design was to compare clinical and immunological end points before and 6 months after
treatment within each dosing group (paired t test) and between groups (2-tailed t test). P < 0.05
was designated as statistically significant. Given the absence of statistically significant group
differences, the data were combined for secondary statistical analysis between the tumor-found
and no-tumor-found subgroups (2-tailed t test). Frequencies were analyzed using Fisher’s exact
test, and correlation analysis utilized Pearson correlations.

Results

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Clinical response (OMS motor severity)

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FLAIR therapy decreased mean total score from 20.1  1.8 (SEM) to 4.1  1.3 in the 1200

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mg/m2 group and from 20.9  1.5 to 5.0  0.7 in the 1500 mg/m2 group (Fig. 1A). In the 1200
mg/m2 group, 5 patients (50%) dropped 1 severity category from moderate to mild; 2 (20%)
dropped from severe to mild. In the 1500 mg/m2 group, 10 (45%) dropped from moderate to
mild, 6 (27%) from severe to mild. All 32 children were in the mild category after treatment.

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Side effects were principally steroidal, such as weight gain, tolerable, and transient in either
treatment group.
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CSF B cell response

The mean frequency of CSF CD19+ B cells fell significantly in both treatment groups (Fig.
1B) from 4.7  0.6% to 0.2  0.1% in the 1200 mg/m2 group and from 5.1  0.7% to 0.2  0.1%
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in the 1500 mg/m2 group (normal mean < 1%). There were no significant differences between
the 1200 mg/m2 and 1500 mg/m2 groups at 0 or 6 months. In the combined dataset (data not
shown), the degree of CSF B cell depletion was comparable in the tumor-found (pre-treatment,
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4.0  0.9%; post-treatment 0.3  0.1%; 92% reduction; P = 0.01) and no-tumor-found group
(pre-treatment, 5.0  0.6%; post-treatment 0.2  0.1%; 96% reduction; P < 0.0001).

Blood B cell depletion and repopulation/reconstitution

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As anticipated, the blood B cell subset dropped dramatically (Fig. 1C) after treatment. In the
1200 mg/m2 group, the mean B cell percentage decreased from 21.7  2.8% to 8.8 ± 3.0% six
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months after treatment (P = 0.001). In the 1500 mg/m2 group, it went from 26.3  1.9% to 9.4 
2.3% (P < 0.0001).
In the combined dataset, rituximab dose (mg/kg) did not correlate with the percentage of
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post-treatment blood B cells at 6 months (r = 0.13, P = 0.56).

Assuming blood B cells had been depleted prior to the second evaluation, then 5 patients
(71%) in the 1200 mg/m2 group met the repopulation criterion of > 1% circulating B cells and 4
(57%) met the criterion of > 5%. Three patients were approaching B cell reconstitution.
In the 1500 mg/m2 group, 14 patients (82%) exhibited > 1% circulating cells and 10 (59%)
with > 5%. Five patients had reconstituted to their previous B cell frequency or were
approaching it. These group patterns were comparable.

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Humoral immunity

Pre-treatment Ig levels did not differ significantly between groups. Serum IgM levels
declined significantly in each group (-72% for 1200 mg/m2 group, P = 0.02; -64% for 1500
mg/m2 group, P < 0.0001). There were no significant changes in serum IgA. The post-treatment
serum IgG concentration was slightly higher in the 1500 mg/m2 group (P = 0.003), but patients
were IVIg-treated.

OMS relapse

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In the 1200 mg/m2 group, 3 patients (30%) relapsed within 6 months of receiving rituximab;

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in the 1500 mg/m2 group, 6 patients (27%). In the second six-month period, one patient (10%)
relapsed in the 1200 mg/m2 group; two in the 1500 mg/m2 group (illness-induced). This brought

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the relapse frequency for one year to 4 of 10 (40%) and 8 of 22 (36%), respectively, which was
not statistically different (P = 1.00, Fisher’s exact test). All but one relapse was mild. Nine
responded to increased ACTH, one to additional rituximab, and two to additional IVIg and
ACTH dose increase.

Discussion US
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This retrospective study yields several useful insights. First, either rituximab dose in FLAIR-
CI induced an impressive and rapid clinical induction in OMS over the complete spectrum of
severities. It is the reduced rituximab induction dose that is new for OMS. Clinical effectiveness
and tolerability seemed comparable, but they must be evaluated in a larger prospective study.
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The optimal rituximab dosing strategy for pediatric OMS, like most other autoimmune disorders,
has yet to be identified. Rituximab dosing will likely undergo many more permutations in an
effort to improve response and prevent relapse. Lower doses reduce the cost of treatment and
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possibly adverse effects, such as serious infections. Rituximab dose reduction for an
inflammatory disorder is not conceptually novel, as it is a subject of ongoing research for various
other disorders, such as multiple sclerosis16 and rheumatoid arthritis in adults.17 Thus, doing so is
not solely the authors’ practice, but supports it.
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Second, reduced-dose rituximab was as effective immunologically against CSF B cell

expansion as the standard higher dosing during the study period. Several lines of evidence now
support the hypothesis that B cells play a critical role in the pathophysiology of OMS: B cell
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expansion and positive oligoclonal bands in the CSF, correlation of motor severity with CSF B
cell subset frequency, reduction or elimination of B cell expansion by rituximab, evidence that
rituximab interrupts B cell trafficking into the brain and that rituximab may remove the B cell as
an antigen-presenting cell because IgG antibodies persist, and B cell activating factor (BAFF)
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and B cell chemoattractant-1 (CXCL13) are elevated in CSF.9

Neither group developed rituximab-induced chronic hypogammaglobulinemia or B
lymphopenia. However, because its frequency is so low, results from 50 – 100 children may be
necessary to determine if lowering the rituximab dose reduces the risk (if dose-dependent), or
whether the reaction is idiosyncratic (dose-independent) or related to other factors. The practice
of ruling out pre-existing hypogammaglobulinemia and periodic monitoring serum
immunoglobulin concentrations and B cell frequency during and after rituximab treatment is
advised.

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Strengths of this study include the combination of clinical and immunological data,
documentation of CSF B cell expansion, video-documentation of neurological response, use of
the same examiner and scorer to assess response, blinded video scoring, use of a validated
response tool, and reasonable exclusion of treatment variables other than rituximab dose. A
limitation of the study was that it was small and retrospective. Our data are preliminary and
cannot address the long-term impact on neurological relapse, morbidity, or safety. The reduced-
dose group was smaller, hence had less statistical power in frequency analyses.
In sum, this retrospective study demonstrated that rituximab dose reduction by 20% did not
diminish the capacity for inducing neurological remission or treating neuroinflammation. Neither

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dose prevented relapse. Though exploratory, and not a clinical trial, this is the first such evidence
and proof-of-concept, encouraging the dose of rituximab to be challenged per disease type. To

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assess whether rituximab dose reduction prevents chronic B lymphopenia warrants a larger,
long-term prospective study.

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Acknowledgments

The National Pediatric Neuroinflammation Organization, Inc., is a Florida charitable

organization and a registered 501(c)(3) organization, of which Dr. Pranzatelli is founder and
president. He is an Adjoint Professor of Neurology at the University of Colorado School of
Medicine, which had no part in this research. The authors thank Lorenda A. Hellman, BS, MT,
ASCP (flow cytometrist at St. John’s Hospital, Department of Flow Cytometry), treating
physicians, and all participating patients and families.

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Funding

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This research did not receive any specific grant from funding agencies in the public, commercial,

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or not-for-profit sectors.

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References

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myoclonus syndrome: immunophenotyping of CSF lymphocytes. Neurology. 2004;62:1526-
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3. Pranzatelli MR, Tate ED, Verhulst SJ, et al. Pediatric dosing of rituximab revisited: serum
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9. Pranzatelli MR, Tate ED. Opsoclonus myoclonus syndrome. In: Swaiman et al. (eds),
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rituximab: first monoclonal antibody approved for the treatment of lymphoma. Semin Oncol.
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TABLE.
Comparative Demographic and Clinical Data at Initial Evaluation
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Clinical parameter Rituximab dosing regimen

1200 mg/m2 1500 mg/m2

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Sample size (n) 10 22
Gender (M:F) 3:7 10:12

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Age at OMS onset (yr) ( SEM) 2.1  0.8 2.2  1.6
OMS duration (yr) 0.5  0.8 0.2  0.2

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Acute 6 (60%) 16 (73%)
Subacute 3 (30%) 5 (23%)

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Chronic 1 (10%) 1 (4%)
OMS Severity (Total Score) 20 ± 5 21 ± 6
Mild 2 (20%) 3 (14%)
Moderate 5 (50%) 12 (54%)
Severe
Tumor
Neuroblastoma†
3 (30%)
3 (30%)
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7 (32%)
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Ganglioneuroblastoma 1 2
History of relapse 5 (50%) 11 (50%)
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One patient Stage 4; the rest, Stage 1. Tumors had been removed prior to treatment.
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Fig. 1. Effect of rituximab dose schedule on (A) Total Score, (B) CSF CD19+ B cell frequency,
and (C) blood CD19+ B cell frequency. Red lines indicate means; error bars are SEM. Statistical
comparisons are indicated by dotted lines for unpaired t tests and by solid lines for paired t tests.
Percent reductions in group means are shown beside the treated groups. (A) Videotapes were
rated by an experienced, blinded scorer (EDT) using the validated OMS Evaluation Scale, a
videotape of which has been published. The sum of each of 12 items scored from 0-3 was
tabulated as ―total score‖ and further designated as ―mild‖ if 0-12, ―moderate‖ if 13-24, or
―severe‖ 25-36.

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