Tomita et al. 1
Supplementary material
Methods for pathological assessment
1. Cytology of the cerebrospinal fluid
The cytology of the cerebrospinal fluid was performed in 28 patients through routine
methods. The sample was centrifuged, and the sediment was collected from at least 1 ml
of cerebrospinal fluid and stained using Papanicolaou and Giemsa methods. At least
two examiners determined the presence of atypical cells.
2. Assessment of sural nerve biopsy specimens
A sural nerve biopsy was performed in 20 patients as previously described (Sobue et al.,
1989; Koike et al., 2003, 2010). The specimens were divided into two portions. The first
portion was fixed in 2.5% glutaraldehyde in 0.125 M cacodylate buffer (pH 7.4) and
embedded in epoxy resin for morphometric and ultrastructural studies. The density of
myelinated fibres was assessed in toluidine-blue-stained semi-thin sections using a
computer-assisted image analyser (Luzex FS; Nikon, Tokyo, Japan), and the density of
small and large myelinated fibres was calculated as previously described (Sobue et al.,
1990a;
Koike
et
al.,
2001,
2004).
For
the
electron
microscopic
study,
epoxy-resin-embedded specimens were cut into ultra-thin transverse sections and
stained with uranyl acetate and lead citrate. To assess the density of unmyelinated
fibres, electron microscopic photographs were taken at a magnification of 4000× in a
random fashion to cover the area of ultra-thin sections, as previously described (Koike
et al., 2003, 2007, 2008b). These electron micrographs were enlarged to 6500×. A
fraction of the glutaraldehyde-fixed sample was processed for a teased-fibre study, and
pathological conditions were microscopically assessed according to the previously
described criteria (Sobue et al., 1989; Dyck et al., 2005). The control values were based
Tomita et al. 2
on a previous report (Koike et al., 2008b).
The second portion of the specimen was fixed in a 10% formalin solution and
embedded in paraffin. Sections were cut by routine methods and stained with
haematoxylin and eosin. Sections were cut by routine methods and stained with
haematoxylin and eosin and Congo red. Immunohistochemical assessments were
performed using the peroxidase-antiperoxidase method in consecutive deparaffinised
sections (Sobue et al., 1990b; Asano et al., 2005, 2006). We assessed the presence of an
atypical cellular appearance of the lymphocytes that infiltrated the peripheral nerve
using haematoxylin-eosin stain. The diagnoses of the types of lymphomas were made
through immunohistochemical studies using antibodies for CD3, CD20, CD45RO,
CD79a, bcl2, Ki67 (DAKO), CD10 (NOVO), and CCR4 (BD biosciences) as
previously described (Asano et al., 2005, 2006). In addition, the presence of
Epstein-Barr virus small ribonucleic acids was examined by in situ hybridisation using
Epstein-Barr
virus-encoded
small
nuclear
early
region
oligonucleotides
on
formalin-fixed, paraffin-embedded sections as previously described (Asano et al.,
2005).
3. Assessment of the autopsied cases
An autopsy was performed in 5 patients (patients 1, 2, 7, 8, and 19) as described
previously (Sobue et al., 1989, 1990a; Koike et al., 2010). Tissues were fixed in a 10%
formalin solution, embedded in paraffin, cut, and stained as described for the sural
nerve specimens. In 2 patients (patients 7 and 8), the median nerve from the axilla to the
wrist and the sciatic/tibial nerve from the upper thigh to above the medial malleolus
were also removed and fixed in 0.05 M phosphate buffer (pH 7.4) containing 1.5%
glutaraldehyde and 3% formalin (Sobue et al., 1990a; Koike et al., 2010, 2011b). After
Tomita et al. 3
fixation, samples were taken every 4 cm along the nerves, embedded in paraffin or
epoxy resin, cut, and examined as described for the sural nerve specimens (Koike et al.,
2004, 2011b). Portions of the ventral and dorsal spinal roots were also fixed and
processed in the same manner.
Representative cases
A
case
with
multiple
mononeuropathy
caused
by
pathologically-proven
neurolymphomatosis (patient 1): A 56-year-old man presented with a 4-month history
of paresthesia and spontaneous pain in the right thigh and weakness in the right lower
limb. The neurological examination revealed weakness, muscle atrophy, and sensory
disturbance in the proximal portion of the right lower limb. Deep tendon reflexes were
absent in the right lower limb. Three months after admission, sensory disturbance
appeared in the ulnar side of the left lower arm. The laboratory investigations revealed
unremarkable findings, with normal lactate dehydrogenase levels (195 IU/l; normal
119-229) and normal soluble interleukin-2 receptor levels (343 U/ml; normal 220-530).
The serum protein immunoelectrophoresis study disclosed no monoclonal gammopathy.
The cerebrospinal fluid showed 1 cell/mm3, with a protein level of 47 mg/dl and no
abnormal cells. The nerve conduction studies showed a reduction in the compound
muscle action potential in the right tibial nerve (2.7 mV) and a conduction block in the
right femoral nerve. Gadolinium-enhanced magnetic resonance imaging (MRI) of the
spinal cord showed no abnormality except for a slight degree of stenosis of the cervical
and lumbar spinal canal. Whole-body gallium-67 scintigraphy was performed, but
abnormal uptake was not noted. A sural nerve biopsy was performed, and the findings
showed axonal degeneration in the teased fibre study (88%) but no infiltration of
lymphomatous cells or demyelination. The patient underwent methylprednisolone pulse
Tomita et al. 4
therapy at 1,000 mg/day for 3 days. His sensory disturbance and spontaneous pain
improved, but his muscle weakness and activity of daily life did not improve. Twelve
months after the neurological onset, a palatal and inguinal tumour appeared. The biopsy
of the palatal tumour revealed a pathological diagnosis of diffuse large B-cell
lymphoma. Chemotherapy was initiated to treat lymphoma, and his spontaneous pain
began to improve thereafter. However, 2 months after chemotherapy, consciousness
disturbance and cardiac arrest occurred suddenly, and he died 14 months after the
neurological onset. An autopsy was performed, and the findings showed invasion of
lymphomatous cells with demyelination in the nerve root and the proximal portion of
the sciatic nerve.
A
case
with
multiple
mononeuropathy
caused
by
FDG-PET-assesed
neurolymphomatosis (patient 10): An 82-year-old woman, who was diagnosed with
diffuse large B-cell lymphoma when she was 76 years old, presented with a five month
history of paresthesia, spontaneous pain, and muscle weakness in the right hand and
arm. Severe muscle weakness was present, and deep tendon reflexes were absent in the
right upper limb. All sensory modalities were impaired in the distal portion of the right
upper limb. The soluble interleukin-2 receptor levels were normal (509 U/ml). The
cerebrospinal fluid showed 1 cell/mm3, with a protein level of 30 mg/dl and no
abnormal cells. The nerve conduction study demonstrated a reduction in the sensory
nerve action potential in the right median nerve (2.8 μV). Fluorodeoxyglucose positron
emission tomography (FDG-PET) imaging showed an increased uptake of FDG along
the right brachial plexus (Fig. 2A). A conduction block was present between the erb and
the axilla, i.e., between the proximal and distal part of the nerve segments with
increased FDG uptake (Fig. 2B), which indicates that segmental demyelination was
Tomita et al. 5
present in the brachial plexus. Left inguinal lymph node and left axillary skin biopsies
were performed, and both revealed CD20-positive atypical lymphocytes. The patient
was diagnosed as having a relapse of lymphoma and received chemotherapy. Her
neurological symptoms improved, and the abnormal finding on FDG-PET and the
conduction block in the nerve conduction study disappeared after treatment.
Lymphomatous invasion at the right brachial plexus was strongly indicated by the
findings of FDG-PET and by the response of her neurological symptoms and FDG-PET
findings to chemotherapy.
A case with paraneoplastic chronic inflammatory demyelinating polyneuropathy
(CIDP)-type features (patient 17): A 61-year-old man developed slowly progressive
paresthesia in his feet. Eight months later, paresthesia in his hands and weakness in the
lower extremities appeared. He was admitted to our hospital 12 months after the
neurological onset. Muscle weakness was present mildly in the upper extremities and
severely in the lower extremities. Vibratory sensitivity of the lower extremities in the
distal portion was absent, and the Romberg sign was positive, although the disturbance
upon superficial sensations was mild. He was not able to walk by himself. The lactate
dehydrogenase and soluble interleukin-2 receptor levels were slightly elevated (282 IU/l
and 635 U/ml, respectively). The serum protein immunoelectrophoresis study disclosed
IgG
λ
monoclonal
gammopathy.
Anti-myelin-associated
glycoprotein
and
anti-sulphoglucuronyl glycosphingolipid antibodies were negative. The cerebrospinal
fluid showed 1 cell/mm3, with a protein level of 191 mg/dl and no abnormal cells.
FDG-PET imaging showed diffuse uptake of FDG in the right iliopsoas muscle. In the
nerve conduction study, motor nerve conduction velocities, distal latencies, and
compound muscle action potentials in the median and ulnar nerves were 14.0 and 20.0
Tomita et al. 6
m/s, 9.1 and 8.9 ms, and 2.9 and 1.2 mV, respectively. The sural nerve biopsy showed
demyelination in the teased fibre study (31%) and axonal degeneration (43%). The
patient became able to walk alone after the IVIg treatment. A bone marrow puncture
was performed, and he received the diagnosis of lymphoplasmacytic lymphoma.
A
case
with
unclassified
multiple
mononeuropathy
caused
by
possible
neurolymphomatosis (patient 21): A 42-year-old man presented with an 8-month history
of hyperesthesia in the left sole and weakness in the left leg. His general physical
examination was unremarkable. The neurological examination revealed bilateral
weakness in the lower legs, predominating on the left side. All sensory modalities were
impaired in the distal portion of the left lower limb. The left Achilles tendon reflex was
absent. The laboratory investigations revealed unremarkable findings. The lactate
dehydrogenase levels were normal (131 IU/l; normal 119-229). The soluble
interleukin-2 receptor levels were slightly elevated (558 U/ml; normal 220-530) but
then became normal on a subsequent examination. Anti-ganglioside and anti-Hu
antibodies were absent. The serum protein immunoelectrophoresis study disclosed no
monoclonal gammopathy. The cerebrospinal fluid was clear with a normal protein level
of 35 mg/dl, and no abnormal cells were observed. The nerve conduction studies
showed a reduction in the compound muscle action potential in the left tibial nerve (0.3
mV) and temporal dispersion in the right tibial nerve. MRI of the head and spinal cord
as well as enhanced CT of the chest, abdomen, and pelvis were normal. Whole-body
gallium-67 scintigraphy was performed, but abnormal uptake was not noted. The patient
underwent a 5-day course of intravenous immunoglobulin (IVIg) therapy (0.4 g/kg/day)
6 times and methylprednisolone pulse therapy at 1,000 mg/day 5 times for 3 days based
on a tentative diagnosis of CIDP. In addition, oral doses of 20 mg/day prednisone and 3
Tomita et al. 7
mg/day tacrolimus were given. Mild and transient improvement was seen each time
after the IVIg and methylprednisolone pulse therapies were performed, but his overall
neuropathic symptoms gradually progressed despite the treatments. Amyotrophy of the
left distal leg and left sciatic neuralgia then appeared. Approximately 1 year after the
first examination, FDG-PET imaging was performed, and increased uptake of FDG in
the right testis was detected. Extirpation of the testicular tumour was performed, and the
pathological diagnosis was diffuse large B-cell lymphoma. Chemotherapy was initiated
to treat the lymphoma, and the neurological symptoms began to improve thereafter.
Neurolymphomatosis was strongly suspected because of the locality of the neurological
symptoms and the presence of malignant lymphoma, although a nerve biopsy was not
performed in this patient.