# Rituximab Administration in Third Trimester of Pregnancy Suppresses Neonatal B-Cell Development

Authors: D. T. Klink; R. M. van Elburg; M. W. J. Schreurs; G. T. J. van Well
Journal: Clinical and Developmental Immunology
 (2008)

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## Abstract

We describe the effect on the neonate of administration of rituximab to a woman with idiopathic thrombocytopenic purpura (ITP). Rituximab, an anti-CD20 antibody, was given weekly for 4 weeks to a woman with ITP in her third trimester of pregnancy. One month after the last rituximab administration a healthy girl was born. She had normal growth and development during the first six months. At birth, B-lymphocytes were not detectable. Rituximab levels in mother and neonate were 24000 and 6700 ng/mL, respectively. Only 7 cases of rituximab administration during pregnancy were described. No adverse events are described for fetus and neonate. We demonstrate that rituximab passes the placenta and inhibits neonatal B-lymphocyte development. However, after 6 months B-lymphocyte levels normalized and vaccination titres after 10 months were adequate. No infection-related complications occurred. Rituximab administration during pregnancy appears to be safe for the child but further studies are warranted.


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## Body

## 1. Introduction

Management of a
pregnant patient with immune-mediated thrombocytopenia (ITP) is similar to that
of nonpregnant patients [1]. Corticosteroids are very
efficient and inexpensive and commonly used for this indication [2]. Although considered
generally safe, steroids have been reported to have adverse effects, including
oral clefts and osteoporosis [3]. Intravenous immunoglobulins
(IVIGs) are a good alternative. In case of refractory ITP in pregnant patients,
a combination of corticosteroids and IVIG can be given. Similar to nonpregnant
patients, a splenectomy can be considered. Remission of ITP is achieved in 70%
of pregnant women after splenectomy [4].
When splenectomy is considered it should be performed in the second trimester of pregnancy. The aim of
treatment of ITP in pregnancy is adequate platelet count to reduce the risk of
hemorrhage during labour. Both mother and neonate are at risk for severe
bleeding as low-neonatal platelet count occurs in 20–25% of the
offspring of ITP patients [5]. When conventional therapy
fails, treatment with third- and fourth-line medications for refractory ITP is
indicated. Unfortunately, their safety during pregnancy has not been
established. For example, azathioprine is associated with intrauterine growth
retardation and immunosupression [6, 7], and medications such as
danazol and vincristin should be avoided due to their teratogenicity [8]. In order to provide safe
alternatives during pregnancy, novel treatment strategies are under
investigation for the management of ITP. These innovative approaches include
thrombopoietin, anti-CD40 ligand, and rituximab, a monoclonal anti-CD20
antibody [1].Rituximab
is a chimeric human and murine monoclonal antibody and targets the CD20 antigen
expressed on pre-B cells and mature B-lymphocytes [9]. Rituximab-opsonized B-cells
are destroyed by at least three pathways: lysis through complement-mediated
cytotoxicity, opsonic phagocytosis, and natural killer cell-mediated
cytotoxicity [10]. The antibody was originally
developed for the treatment of B-cell lymphoma [11]. Recently, however, the use
of rituximab is expanded to the treatment of different autoimmune diseases
including ITP [12]. The number of
(autoreactive) B-cells is diminished in patients treated with rituximab. Since
rituximab is an IgG-based antibody, it may cross the placenta and then
interfere with fetal and neonatal B-cell development, potentially leading to
immune deficiency and increased susceptibility to infections in the neonate.
However, few data are available on the use of rituximab during pregnancy.We
present a case report on the effects of rituximab on a child after exposure
during third trimester pregnancy. In addition, we performed a literature search
for additional data in order to gain more insight on the safety aspects of
rituximab administration during pregnancy.Case
A 36-year-old
woman was diagnosed with ITP since 1994. At that time during pregnancy, ITP was
successfully treated with corticosteroids. In 1997, a splenectomy was
performed. During present pregnancy, she was initially treated with
corticosteroids and IVIG, but the platelet count only rose from 16 × 109/L
to 27 × 109/L with persisting hemorrhagic diathesis.Subsequently, she was referred to our
hospital and treated for refractory ITP with 60 mg  prednisone daily. This treatment was continued during pregnancy until two weeks
prior to delivery. In addition, from week 30 to week 34, she also received
rituximab 375 mg/m2 intravenously weekly for 4 consecutive weeks.
Due to persisting low-platelet count, rituximab administration was ceased and
IVIG administration was started for two consecutive days at 37 weeks and 6 days
of gestation. At 38 weeks and 2 days of gestation and one month after the last
rituximab administration, labour was induced with prostaglandins. For comfort,
the mother received promethazine orally and pethidine intravenously until one
hour before birth. A slightly depressed girl was born with apgar scores of 7,
8, and 8 at 1, 5, and 10 minutes, respectively. Birth weight was 3780 grams (90th–95th percentile
weight for gestational age according to Kloosterman [13]), length was 46 centimetre (cm) (third percentile length for gestational age [14]), and head circumference was 36 cm (98th percentile head
circumference for gestational age [14]). Physical examination
revealed no abnormalities. Cord blood samples were taken for blood gas analyses
and measurement of thrombocytes, lymphocyte characterization, and rituximab
levels. Because of the administration of opiates intravenously prior to birth,
the slightly depressed neonate was subsequently admitted to the high-care
facility of our neonatology department for observation. In the next 24 hours,
she quickly recovered spontaneously and no adverse events occurred. The patient
was transferred to the maternity ward and discharged on the fourth day after
birth. She was followed up in the outpatient clinic for pediatric infectious
diseases and immunology of our hospital. Up to the age of 10 months, she
developed normally, and growth was not impaired. She did not have any
infectious complications. Vaccination titres were normal.


## 2. Methods

### 2.1. Rituximab Serum Level Measurement

The rituximab
serum levels in cord blood were measured using enzyme-linked immunosorbent
assay (ELISA) at Xendo Drug Development, Groningen, The Netherlands. Coefficient of variation was between 4.7% and 8.2%.


### 2.2. Flow Cytometry

Lymphocyte
characterization was performed at the Department of Pathology. For this
purpose, whole blood was incubated in Trucount tubes with fluorochrome-conjugated
monoclonal antibodies directed against lymphocyte associated markers CD3, CD4,
CD8, CD16/56, CD19, CD20, CD27, and CD45 (BD Biosciences, San Jose CA). After
red cell lysis, lymphocyte subsets were analyzed by multiparameter flow
cytometry (FACSCalibur, BD Biosciences). B-lymphocytes were quantified as the
fraction of CD19 positive cells within the CD45 positive total lymphocyte gate.
B-lymphocyte maturation and memory formation were analyzed as the percentage of
CD19 and CD20 positive, and CD19 and CD27 positive cells within the total
lymphocyte gate, respectively.


### 2.3. Review of the Literature

Two Embase.com
searches were performed on May 1, 2007, with EMTree-terms: (1) (“rituximab”/exp/dd_ae (adverse drug reaction), dd_it (drug interaction), dd_to (drug toxicity) AND (“newborn”/exp OR “infant”/exp OR “fetus”/exp OR “embryo”/exp OR “pregnancy”/exp))
which resulted in 19 references; (2) (“rituximab”/exp
AND “pregnancy”/exp)—54 references.
Duplicate references from both searches were removed, resulting in 42
references. Titles and abstracts identified were examined to select potentially
relevant studies. In addition, reference lists of identified studies and review
articles were examined.


## 2.1. Rituximab Serum Level Measurement

The rituximab
serum levels in cord blood were measured using enzyme-linked immunosorbent
assay (ELISA) at Xendo Drug Development, Groningen, The Netherlands. Coefficient of variation was between 4.7% and 8.2%.


## 2.2. Flow Cytometry

Lymphocyte
characterization was performed at the Department of Pathology. For this
purpose, whole blood was incubated in Trucount tubes with fluorochrome-conjugated
monoclonal antibodies directed against lymphocyte associated markers CD3, CD4,
CD8, CD16/56, CD19, CD20, CD27, and CD45 (BD Biosciences, San Jose CA). After
red cell lysis, lymphocyte subsets were analyzed by multiparameter flow
cytometry (FACSCalibur, BD Biosciences). B-lymphocytes were quantified as the
fraction of CD19 positive cells within the CD45 positive total lymphocyte gate.
B-lymphocyte maturation and memory formation were analyzed as the percentage of
CD19 and CD20 positive, and CD19 and CD27 positive cells within the total
lymphocyte gate, respectively.


## 2.3. Review of the Literature

Two Embase.com
searches were performed on May 1, 2007, with EMTree-terms: (1) (“rituximab”/exp/dd_ae (adverse drug reaction), dd_it (drug interaction), dd_to (drug toxicity) AND (“newborn”/exp OR “infant”/exp OR “fetus”/exp OR “embryo”/exp OR “pregnancy”/exp))
which resulted in 19 references; (2) (“rituximab”/exp
AND “pregnancy”/exp)—54 references.
Duplicate references from both searches were removed, resulting in 42
references. Titles and abstracts identified were examined to select potentially
relevant studies. In addition, reference lists of identified studies and review
articles were examined.


## 3. Results of Reported Case and Literature Search

Our literature search resulted in 7
additional case reports on rituximab use during pregnancy. The relevant data of
8 cases, including our case, are summarized in Table1.Table 1
Summary literature search.
MotherHerold 2001Kimby 2004Friedrichs 2006Scully M 2006Ojeda-Uribe 2006Magloire 2006Decker 2006Current studyConditionB-cell lymphomaNH-lymphomaBurkitt lymphomaThrombotic
Thrombocytopenic PurpuraAutoimmune Haemolytic anaemiaBurkitt lymphomaB-cell-NH lymphomaITPRituximab 375 mg/m2Weekly 4xWeekly 4xWeekly  4xWeekly 4x——Biweekly 6xWeekly 4XCo medicationCHOP(1)—CHOPPlasma exchangeCorticosteroidsCHOPCHOPPrednisone, IgGAdministration time GAWeek 21Week -1 to 3Weeks 16–30Week 27Week 10Week 13 to ?Weeks 16-28Week 30 to 34Rituximab level D0(2)(ng/mL)——9750———2500024000CD19+ B-cells D0 (1 × 109/L)——0———00Child————————GA (weeks)3540413038393338Rituximab level D0
(ng/mL)——32095———300006700CD19+ B-cells (1 × 109/L) < week 1 post partum—0,10—0,66—approx 0,050,08CD19+ B-cells (1 × 109/L)
1 monthnormal———1,98——0,00CD19+ B-cells (1 × 109/L) 3 months———————0,21CD19+ B-cells (1 × 109/L)
6 months———————2Vaccination titres—NormalNormal———NormalnormalIgA; IgM; IgG (g/L) 1-2 months————0,07; 0,3; 4,5-——<0,07; 0,05; 6,1IgA; IgM; IgG (g/L)
3 months———————<0,07; <0,04; 2,8IgA; IgM; IgG (g/L) ≥6 months—IgG normalNormal———Normal0,16; 0,53; 2,2(1) Cyclophosphamid,
doxorubicin, vincristin, prednisolon.(2) Day of birth.


### 3.1. Rituximab Levels and B-Lymphocytes in the Mother

In the majority of the cases, the
indication for rituximab was the presence of a B-cell malignancy. In 3 cases,
including our case, the indication was a haematological disorder which involved
the immune system. Regardless of the indication, the dosage was the same in all
cases with a frequency of either once a week or every 14 days. Repetition of
dosage was either 4 or 6 times. In combination with nonalkylating chemotherapy
(CHOP), rituximab was effective in reducing tumorload in 5 cases (Table1). In
2 cases [15, 16] and our case, maternal B-cell numbers were determined at the day of
birth and were not detectable. Decker et al. [15] reported a reconstitution of maternal B-cells 12 weeks after
delivery coinciding with a large decrease in serum concentration of rituximab.
At birth, the maternal rituximab levels were reported to be approximately 25 mg/L when administrated a month prior to delivery. We have found equal
rituximab levels in our case. Friedrichs et al. [16] reported a lower level of 9.7 mg/L, but in this case, report
rituximab was administrated around 12 weeks prior to delivery. When followed up,
the rituximab levels decreased corresponding with its known half-life time [15, 16].


### 3.2. Rituximab Levels and B-Lymphocytes in the Neonate

At birth, the
neonatal rituximab level in our case was 6.7 mg/L. Two other authors reported
neonatal rituximab levels at birth: Friedrichs et al., and Decker et al.
reported a level of 32 mg/L and 30 mg/L, respectively. Both authors reported a
decline in rituximab levels consistent with the known half-life time of
rituximab.In 2
cases [15, 16] and our case, the number of
neonatal B-cells at time of delivery was determined and was undetectable by
flow cytometry. Time of rituximab administration during gestation did not
appear to influence this outcome. In our case, the number of B-lymphocytes
demonstrated a steady increase over time. Although the number of B-cells was
still zero 3 weeks after birth, a rise was detected after 3 months. At the age
of 6 months, the number of B-cells was in the normal range both in absolute and
in relative numbers. Moreover, at this time, all peripheral B-cells were mature
as indicated by CD20 positivity, and B-cell memory formation could be
demonstrated by CD27 positivity (Figure 1). In addition, normal immunoglobulin
(IgG) levels could be demonstrated which is consistent with findings in other
case reports. In our case and in 3 other cases, [15–17] normal vaccination responses
were found.Flow cytometric analysis of
peripheral blood lymphocytes, taken from the neonate at the age of six months,
indicating the presence of CD19 positive B-lymphocytes (20% on average; (a) and (b)),
expressing the maturation marker CD20 (99%; (a), and partly expressing the
memory marker CD27 (7%; (b). Analysis was done within the CD45 positive total
lymphocyte gate.
(a)(b)


### 3.3. Clinical Outcome of the Neonate

During
pregnancy, fetal growth and development were monitored and turned out to be
normal. Indeed, in our case, the patient’s birth weight was between the 90th
and 95th percentile [13], within the normal range of
birth weight in the Dutch population (outer limit is 97th percentile).Five
out of eight neonates were born at term age (Table1). Of the three preterm
neonates, one was delivered by caesarean section due to deterioration of the
maternal condition [18]. No apparent cause for the
preterm birth was reported in the other two neonates. Only one neonate [19] was described to have been
treated with antibiotics for suspicion of perinatal infection. All other
children did not have any complications in the neonatal period. More
importantly, no single adverse event occurred.In our
case, the neonate was admitted to the high-care facility for observation due to
maternal intravenously opiates administration. The arterial umbilical cord
blood gas analysis was pH 7.26, CO2 57.0 mmHg, 25.0 HCO3− mmol/L, and base excess −3.1 mmol/L, indicating little intrauterine hypoxic
stress. Although our patient was slightly depressed at birth, she quickly
recovered. Thus, the need for further blood gas analysis was eliminated.All
the children had normal growth and development. No clinical signs of impaired immunity
were observed.


## 3.1. Rituximab Levels and B-Lymphocytes in the Mother

In the majority of the cases, the
indication for rituximab was the presence of a B-cell malignancy. In 3 cases,
including our case, the indication was a haematological disorder which involved
the immune system. Regardless of the indication, the dosage was the same in all
cases with a frequency of either once a week or every 14 days. Repetition of
dosage was either 4 or 6 times. In combination with nonalkylating chemotherapy
(CHOP), rituximab was effective in reducing tumorload in 5 cases (Table1). In
2 cases [15, 16] and our case, maternal B-cell numbers were determined at the day of
birth and were not detectable. Decker et al. [15] reported a reconstitution of maternal B-cells 12 weeks after
delivery coinciding with a large decrease in serum concentration of rituximab.
At birth, the maternal rituximab levels were reported to be approximately 25 mg/L when administrated a month prior to delivery. We have found equal
rituximab levels in our case. Friedrichs et al. [16] reported a lower level of 9.7 mg/L, but in this case, report
rituximab was administrated around 12 weeks prior to delivery. When followed up,
the rituximab levels decreased corresponding with its known half-life time [15, 16].


## 3.2. Rituximab Levels and B-Lymphocytes in the Neonate

At birth, the
neonatal rituximab level in our case was 6.7 mg/L. Two other authors reported
neonatal rituximab levels at birth: Friedrichs et al., and Decker et al.
reported a level of 32 mg/L and 30 mg/L, respectively. Both authors reported a
decline in rituximab levels consistent with the known half-life time of
rituximab.In 2
cases [15, 16] and our case, the number of
neonatal B-cells at time of delivery was determined and was undetectable by
flow cytometry. Time of rituximab administration during gestation did not
appear to influence this outcome. In our case, the number of B-lymphocytes
demonstrated a steady increase over time. Although the number of B-cells was
still zero 3 weeks after birth, a rise was detected after 3 months. At the age
of 6 months, the number of B-cells was in the normal range both in absolute and
in relative numbers. Moreover, at this time, all peripheral B-cells were mature
as indicated by CD20 positivity, and B-cell memory formation could be
demonstrated by CD27 positivity (Figure 1). In addition, normal immunoglobulin
(IgG) levels could be demonstrated which is consistent with findings in other
case reports. In our case and in 3 other cases, [15–17] normal vaccination responses
were found.Flow cytometric analysis of
peripheral blood lymphocytes, taken from the neonate at the age of six months,
indicating the presence of CD19 positive B-lymphocytes (20% on average; (a) and (b)),
expressing the maturation marker CD20 (99%; (a), and partly expressing the
memory marker CD27 (7%; (b). Analysis was done within the CD45 positive total
lymphocyte gate.
(a)(b)


## 3.3. Clinical Outcome of the Neonate

During
pregnancy, fetal growth and development were monitored and turned out to be
normal. Indeed, in our case, the patient’s birth weight was between the 90th
and 95th percentile [13], within the normal range of
birth weight in the Dutch population (outer limit is 97th percentile).Five
out of eight neonates were born at term age (Table1). Of the three preterm
neonates, one was delivered by caesarean section due to deterioration of the
maternal condition [18]. No apparent cause for the
preterm birth was reported in the other two neonates. Only one neonate [19] was described to have been
treated with antibiotics for suspicion of perinatal infection. All other
children did not have any complications in the neonatal period. More
importantly, no single adverse event occurred.In our
case, the neonate was admitted to the high-care facility for observation due to
maternal intravenously opiates administration. The arterial umbilical cord
blood gas analysis was pH 7.26, CO2 57.0 mmHg, 25.0 HCO3− mmol/L, and base excess −3.1 mmol/L, indicating little intrauterine hypoxic
stress. Although our patient was slightly depressed at birth, she quickly
recovered. Thus, the need for further blood gas analysis was eliminated.All
the children had normal growth and development. No clinical signs of impaired immunity
were observed.


## 4. Discussion

Rituximab is generally considered safe for the treatment of both
malignant and nonmalignant diseases. Although frequently used in women who are
fertile, little is known about the safety aspects during pregnancy. Since
rituximab is a chimeric antibody of the IgG isotype, it is likely to cross the
placental barrier, and one should consider the risk of B-cell depletion in the
child. To our knowledge, in vitro
studies on placental transfer of rituximab have not been reported, but
rituximab levels could be demonstrated in the exposed neonate. In our case, rituximab
levels were 3.5 times lower in the neonate at birth when compared to the serum
concentrations of the mother. This is in contrast with previously reported
cases [15, 16] in which the rituximab
levels in the cord blood were higher compared to maternal levels. As rituximab
was administrated in the second trimester in these cases, time of administration
during the pregnancy may contribute to this difference. Maternal IgG is
transferred across the placenta by means of a specific receptor-mediated
mechanism. Mother-to-fetus IgG transfer starts at week 16 of gestation. After
22 weeks of gestation, the fetal IgG levels increase rapidly [20]. Thus, when rituximab
exposure starts at gestational age 16 weeks, as occurred in the two cases
reported  [15, 16], there has been a maximum
period of opportunity for transfer. In our case, exposure was relatively short
prior to birth. Although most of IgG is acquired in the last 4 weeks of
pregnancy, the exposure may not have been long enough to reach the levels
previously reported. Despite the lower level of rituximab in the cord blood,
the number of neonatal B-lymphocytes at birth was zero. This is similar to
previous reports [15, 16, 18, 19]. During follow-up, all
children showed an increase of B-lymphocytes to the age-specific normal reference
levels demonstrating the transient effect of rituximab (Table 1).
Reconstitution of B-cells coincided with the known half-life of rituximab.
Although B-cell function was initially impaired, the clinical outcome of all
patients was good. None of the patients had serious infection-related
complications. The explanation for this finding may be the fact that in the
first few months of life, the neonate largely depends on maternal IgG for
immunity, and B-cell function still has to develop. The rituximab effect may
already be diminished properly before the infant depends on its own B-cell
function. The data of adequate IgG and vaccination titres are supportive of
this.Although
the therapeutic benefit of rituximab in malignancies is well established, its
efficacy in the treatment of autoimmune diseases is still under investigation.
Likewise, the exact mechanism(s) by which the B-cell depletion promoted by
rituximab ameliorates autoimmune disease activity remains unclear. Despite lack
of complete understanding of how rituximab targets autoimmune diseases, the
antibody has been applied in the treatment of various ailments such as
rheumatoid arthritis (RA), Systemic Lupus Erythematosus [21, 22], and ITP [12]. Early success with
rituximab in ITP has led to its widespread use and incorporation into recent
treatment schemes. A meta-analysis by Arnold et al. demonstrated [23] that rituximab was
associated with a platelet count response defined as > 50 × 109 cells/L.
However, none of the identified studies included a control group and none met
all predetermined methodological quality criteria for observational studies. The
efficacy of rituximab compared with standard treatments for ITP could not be
determined, and the authors strongly urge for randomized controlled trials.In our
case, rituximab administration did not result in increased platelet count and
rituximab was stopped and IVIG treatment was started. This finding underlines
the need for additional data to determine the effectiveness of rituximab for
ITP in pregnant women.


## 5. Conclusion

Intrauterine exposure to rituximab appears not to be
harmful to the neonate as neither in the literature nor in our case any
complications related to rituximab are reported. However, data are limited to a
handful of case reports, and caution should be taken when considering rituximab
administration during pregnancy. In addition, rituximab should not be used
indiscriminately, but should be reserved for carefully selected cases when
first and second line therapy fails. Further studies are warranted to fully
evaluate the safety of rituximab during pregnancy.



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*Source: 271363-2008-06-25.xml*