Neuropathology 2018

doi:10.1111/neup.12526

Case Report

An autopsy case of progressive multifocal
leukoencephalopathy after rituximab therapy
for malignant lymphoma
Reiji Muto,1,2

Yasuo Sugita,1

Seiya Momosaki,3 Yuriko Ito,4 Yoshiyuki Wakugawa4 and
Koichi Ohshima1

1

Department of Pathology, Kurume University School of Medicine, Kurume, 2Department of Pathology, National
Hospital Organization, Kumamoto Medical Center, Kumamoto, Departments of 3Pathology and 4Cerebrovascular
Disease and Clinical Research Institute, National Hospital Organization, Kyushu Medical Center, Fukuoka, Japan

Progressive multifocal leukoencephalopathy (PML) is a
rare fatal demyelinating disease of the central nervous
system caused by reactivation of the JC virus (JCV),
which is named after the initials of the patient from
whom the virus was first isolated. JCV is highly prevalent worldwide, infects humans in early childhood, and
the infection persists throughout the course of life in
latent form. The present paper deals with the second
autopsy case report of rituximab-associated PML in
Japan. A 63-year-old woman who had undergone chemotherapy for non-Hodgkin lymphoma developed progressive dysarthria and cerebellar ataxia. Head magnetic
resonance imaging (MRI) revealed small, scattered,
hyperintense areas in the midbrain, pons and thalamus,
and the patient was first diagnosed as having cerebral
infarction. Follow-up MRI showed tendency toward cerebellar atrophy and multiple system atrophy cerebellar type
was suggested, which we concluded must have coincidentally occurred. It was challenging to perform biopsy due to
the location of the foci and the patient’s condition. Twelve
months later she died of aspiration pneumonia caused by
the bulbar lesion. At autopsy, the histological examination
suggested the presence of demyelinating foci with numerous foamy macrophages. In the foci, oligodendrocytes with
enlarged ground-glass like nuclei were found in a scattered
manner and astrocytes with bizarre nuclei were also
detected. These findings verified the case as PML. The
first diagnosis of cerebral infarction was later withdrawn,

Correspondence: Reiji Muto, MD, Department of Pathology, School
of Medicine, Kurume University, Asahi-machi 67, Kurume-city, Fukuoka 830-0011, Japan. Email: sugarless610@hotmail.com
Received 27 February 2018; Revised 21 October 2018; Accepted 22
October 2018.

© 2018 Japanese Society of Neuropathology

although appropriate disorders were not recalled even
after testing with various antibodies. The rate of PML
development tends to increase after treatment with
molecular-targeted therapies, which directly or indirectly
attenuate the cellular-mediated immune system. Various
novel molecular-targeted and immunosuppressive drugs
have been released on the market; the cases of PML have
consequently increased. Accordingly, pathologists should
keep this disease in mind in the differential diagnosis
when neural symptoms newly emerge in patients who are
treated with these drugs.
Key words: demyelinating diseases, JC virus, leukoencephalopathy, progressive multifocal, rituximab.

INTRODUCTION
Progressive multifocal leukoencephalopathy (PML) is a
rare demyelinating disorder of the central nervous system (CNS) caused by opportunistic reactivation of the JC
virus (JCV), which is named after the initials of the
patient from whom the virus was first isolated.1,2 JCV is
prevalent worldwide but its seropositivity differs among
countries, with approximately 70% of adults reportedly
being seropositive in Japan.3,4 PML has a high mortality
rate. The mortality rate of human immunodeficiency
virus (HIV)-negative patients with PML after rituximab
therapy is reportedly as high as 90%.5 Recent studies
have confirmed that molecular-targeted and/or immunosuppressant drugs are potential causative agents of this
disease.6–8 Given the widening indication of these drugs
and the increasing frequency with which these medications are being used, the frequency of PML has increased
in recent years.8
Here, we present a rare autopsy case of rituximabassociated PML.

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R Muto et al.

CASE PRESENTATION
Clinical summary
A 63-year-old woman was diagnosed with malignant lymphoma and had started therapy with bendamustine and
rituximab approximately 12 months before death. Eight
months before death, the last dose of rituximab was
administered. Thereafter, the disease had remained in
complete remission.
Approximately 6 months before death, the patient
developed dysarthria and cerebellar ataxia. The cerebrospinal fluid (CSF) was clear with cells 3/mm3 (100% small
mononuclear), and contained 35 mg/dL protein and
57 mg/dL glucose. No organisms were found in the CSF
including cryptococcus. Head magnetic resonance imaging
(MRI) revealed small, scattered, hyperintense areas in the
midbrain, pons, medula oblongata and thalamus, and the
patient was first diagnosed with cerebral infarction, which
was later discounted (Fig. 1A–C). Follow-up imaging
showed tendency toward cerebellar atrophy and hot cross
bun sign in the pontine base area (Fig. 1D). The clinicians
wondered whether the cerebellar atrophy and the mass
lesion in the brain were signs of a single disease or of separate diseases. They concluded that two separate diseases
merged because the patient’s condition could not be

explained by a single disease. They considered the possibility of a drug-induced condition or a para-neoplastic syndrome, but there were no findings or data suggesting such
potentialities. They concluded that a degenerative disease
such as multiple system atrophy cerebellar type (MSA-C),
which was suggested by imaging, coincidentally occurred.
However, even if this was a case of MSA-C, they judged
that the progress of atrophy was fast and that possibly
another disease was involved.
Biopsy of the high intensity lesions was scheduled, but
it was difficult to perform the maneuver due to difficulties
in accessing the site of the foci and the patient’s condition,
which was carefully monitored. Subsequently, the scheduled head MRI revealed tendency toward aggravation.
MSA-C was clinically suspected. However, other diseases
(i.e., invasion and/or metastasis of malignant lymphoma,
paraneoplastic syndrome, demyelinating disease, HIVassociated encephalopathy, and drug-induced encephalopathy) were also considered in the differential diagnosis
due to the condition’s rapid progression. Various antibody
tests, including anti-Hu, anti-Yo, and anti-Ri antibodies,
were negative, as were the intrathecal oligoclonal band
and myelin basic protein; no increase was observed in
interleukin-6 and the immunoglobulin G index. Since it
was difficult to perform a biopsy, only the scheduled MRI

Fig. 1 MRI findings on fluid-attenuated inversion recovery (A, B), T1weighted (C) and T2-weighted (D)
images. (A, B) High intensity areas
are observed in the subcortical lesion
(A) and the left thalamus. (C) A
plaque-like lesion can been seen in
the medulla oblongata. (D) Hot
cross bun sign is found in the pontine
base.

© 2018 Japanese Society of Neuropathology

PML after rituximab therapy

3

Fig. 2 Histopathological findings in the demyelinating lesions. (A) Rarefaction of neural cells and emergence of numerous gemistocytic
astrocytes are seen. (B) Demyelinating foci are initially characterized by oligodendrocytes containing diffuse basophilic ground glass-like
nuclei. (C) Aggregation of foamy macrophages can also be observed. (D) Astrocytes with bizarre nuclei can be seen. (E) In the demyelinating foci, oligodendrocytes with diffuse basophilic inclusion bodies are present as diagnostic hallmarks of PML. (F) Cells with dot-like
inclusion bodies as well as diffuse homogenous ground glass-like nuclei (inset) show JC virus VP-1 protein immunoreactivity. HE (A,
C-E), KB (B), immunohistochemistry (F). Scale bars: 200 μm (A, B), 100 μm (C, F), 50 μm (D, E).

© 2018 Japanese Society of Neuropathology

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R Muto et al.

was performed. Almost 1 year later, dysphagia, probably
caused by worsening of the bulbar lesion, progressively
developed. Repeated aspiration pneumonia led to disseminated intravascular coagulation, and ultimately the
patient’s cause of death was respiratory failure.

Pathological findings
The weight of the brain was 1290 g and age-appropriate.
Although the MRI suggested cerebellar atrophy, no sign
of obsolescence was found in gross pathology. The crosssections showed small discolored lesions in the cerebellum
and the subcortical areas.
Microscopically, multiple small foci of demyelinating
lesions were observed scattered in the medulla, midbrain,
thalamus and cerebellum on sections stained with hematoxylin-eosin (HE) (Fig. 2A). Klüver-Barrera (KB)
staining showed near-total loss of myelin, and demyelinating foci were characterized by initially oligodendocytes
containing diffuse basophilic ground glass-like nuclei
(Fig. 2B). Aggregation of macrophages was seen around
the demyelinating area (Fig. 2C). Reactive hypertrophic
astrocytes with bizarre nuclei were observed (Fig. 2D). In
the demyelinated foci oligodendrocytes with diffuse and
basophilic inclusion bodies are recognized as diagnostic
hallmarks of PML (Fig. 2E). Gliosis was prominent and
numerous macrophages accumulated in the lesions. Cells
with dot-like inclusion bodies as well as diffuse homogenous ground glass-like nuclei (inset) show positive immunoreactivity for the antibody against JCV VP-1 protein
(VP-1, dilution 1:100; Dako Japan, Kyoto, Japan)
(Fig. 2F). These findings are compatible with the presentation of PML.

DISCUSSION
Based on our literature review, nine autopsy cases have
been reported from Japan in English in the past
30 years,9–17 and the present paper is the second autopsy
case report of rituximab-associated PML in Japan
(Table 1).17
The key diagnostic factors that can distinguish PML
from the disorders previously mentioned, are predisposition to the disease and the histological findings.
Any form of immunodeficiency may predispose to
PML.18 Various other underlying diseases and conditions,
including HIV, hematological malignancies, human T-cell
lymphotrophic virus (HTLV)-1 infection, sarcoidosis, and
collagenous diseases have been reported so far.19–23 In
addition,
immunomodulatory
therapies
including
rituximab, which is a monoclonal antibody against the protein CD20, are increasingly being considered as potent
causative agents of this disease.8
In addition, drugs prescribed for multiple sclerosis
including natalizumab, fingolimod, and diethyl fumarate
(DMF) reportedly increase the risk of PML. Natalizumab
inhibits T-cell migration and extravasation.3 Fingolimod, a
sphingosine-1-phosphate receptor modulator, sequestrates
lymphocytes in lymph nodes, and lymphocytes that can be
recruited in the CNS decrease.24 DMF, the precise mechanism of which is unknown, can lead to long-lasting, severe
lymphocytopenia, and DMF-related PML cases have been
reported.25,26
In histological findings, multiple foci are found
scattered in white matter within junctional lesions between
the white and gray matters. In a typical case, myelin disappears, while axons are preserved. Aggregation of

Table 1 Reported autopsy cases of progressive multifocal leukoencephalopathy in the past 30 years
Authors

Age/sex

Predispositions and comorbidities

Brain
weight (g)

Location of lesions

Irie et al. (1992)9
Shintaku et al.. (2000)10

64/F
21/M

CRF (HD)
Common variable immunodeficiency

1100
1340

Yogo et al. (2001)11
Arai et al. (2002)12

34/M
74/F

Wiskott-Aldrich syndrome
CRF (never HD)

Not stated
970

Matsuda et al. (2007)13

57/M

Not stated

Oshima et al. (2009)14

58/M

Takeda et al. (2009)15

64/M

CRF (HD), LC (hepatitis C), and
HTLV-1 infection
Schizophrenia, MDS, chronic
pancreatitis
No underlying diseases

Akiyama et al. (2010)16
Sano et al (2015)17

64/M
66/M

Multiple myeloma
Malignant lymphoma with rituximab
therapy

Not stated
1170

Cerebellum and brain stem
Cerebrum, basal ganglia, thalamus,
brainstem, cerebellum, and spinal cord
Cerebrum, cerebellum and spinal cord
Cerebrum, cerebellum, brain stem
(especially in the pons and medulla
oblongata)
Cerebrum (occipital and temporal
lobes)
Corticomedullary junction of the
temporal, occipital
Cerebrum, mid-brain, pons, medulla
oblongata, cerebellum, spinal cord
(cervical to sacral segments)
Cerebrum (frontal)
Cerebrum, brain stem, cerebellum

1365
1150

CRF, chronic renal failure; HD, hemodialysis; HTLV-1, human T-cell lymphotrophic virus type 1; LC, liver cirrhosis; MDS, myelodysplastic
syndrome.

© 2018 Japanese Society of Neuropathology

PML after rituximab therapy
macrophages is observed in the active foci. The oligodendrocytes in the peripheral lesions with enlarged nuclei,
which are filled with monotonous basophilic material, are
compatible with JCV DNA. Astrocytes with bizarre nuclei
and vast eosinophilic cytoplasms have also been seen.
Finally, it could be concluded when pathologists
encounter unidentified demyelinating lesions in patients
with decreased or defective cell-mediated immunity, they
should include PML in the differential diagnosis and
observe the lesions carefully to confirm the cells with
inclusion bodies.

ACKNOWLEDGMENTS
Not applicable.

DISCLOSURE
The authors have no competing interests or funding to
declare.

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© 2018 Japanese Society of Neuropathology