NEUROLOGY MS ID#: NEUROLOGY/2015/713974Tsujikawa et al.

Supplementary Data

HMGCR antibody-associated myopathy as a paraneoplastic manifestation of

esophageal carcinoma

Koyo Tsujikawa MD1, Kazuhiro Hara MD, PhD 1, Yoshinao Muro MD, PhD 2, Hirotaka

Nakanishi MD, PhD 1, Yukiko Niwa MD, PhD3, Masahiko Koike MD, PhD3, Seiya

Noda MD 1, Yuichi Riku MD, PhD 1, Kentaro Sahashi MD, PhD 1, Naoki Atsuta MD,

PhD 1, Mizuki Ito MD, PhD 1, Yoshie Shimoyama, MD, PhD4, Masashi Akiyama, MD,

PhD 2, Masahisa Katsuno MD, PhD 1.

1) Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya,

Japan.

2) Department of Dermatology, Nagoya University Graduate School of Medicine,

Nagoya, Japan.

3) Department of Gastroenterological Surgery (Surgery II), Nagoya University Graduate

School of Medicine, Nagoya, Japan.

4) Department of Pathology and Laboratory Medicine/Diagnostic Pathology, Nagoya

University Graduate School of Medicine, Nagoya, Japan.

Contents Appendix e-1

e-Reference

Figure e-1, e-2

NEUROLOGY MS ID#: NEUROLOGY/2015/713974Tsujikawa et al.

Appendix e-1

Anti-HMGCR antibodies measured by ELISA. Anti-HMGCR antibodies were

measured with antigen-capture ELISA using the methods we previously described [e1].

Briefly, a 96-well Immobilizer Streptavidin Plate (Thermo Scientific Nunc) was

incubated with 1 μl/well of TnT reaction mixture including biotinylated recombinant

protein derived from the full-length cDNA (#MHS 6278-202801490, GE Healthcare).

Wells were then incubated with sera (1:1,000 dilution) and probed with horseradish

peroxidase-conjugated anti-human IgG antibody (1:30,000, Dako). After incubation

with SuperSignal ELISA Femto Maximum Sensitivity Substrate (Thermo Scientific

Pierce), relative luminescence units (RLU) were determined using a GloMax-Multi

Detection System (Promega). Each serum sample was tested in duplicate, and the mean

RLU was used for data analysis. The RLU of the samples was converted into units using

a standard curve created with a prototype positive serum. The cut-off level of anti-

HMGCR antibody was set at 4.3 units, based on five standard deviations above the

mean value obtained from 26 healthy control sera.

Histopathological examination. The pathological examination was approved by the

Ethics Committee of the Nagoya University Graduate School of Medical Science and

the patient provided written informed consent. We compared HMGCR expression in the

patient’s muscle tissue (MB15-35) with those in control subjects: MB15-15 of

myopathy with anti-mitochondrial antibody, MB15-23 of myopathy with anti-signal

recognition particle antibody, and MB15-54 of myopathy with anti-Jo-1 antibody. We

confirmed that the control subjects were negative for serum anti-HMGCR antibodies.

Muscle biopsy specimens were obtained using open biopsies and were snap-

frozen in isopentane, chilled in dry ice and preserved at -80°C until further use. The

NEUROLOGY MS ID#: NEUROLOGY/2015/713974Tsujikawa et al.

samples were cut according to routine methods into 10-μm cryostat sections and stained

with hematoxylin and eosin. Cryostat sections were fixed in cold acetone, air dried, and

incubated with the following primary antibodies at 4 °C overnight: HLA-ABC / MHC

class I (1:800, BD), HLA-DR / MHC class II (1:300, BD), CD4 (1:150; Dako), CD8

(1:150; Dako), CD68 (1:300; Dako), and HMGCR (1:400; Santa Cruz Biotechnology).

Immunostaining was performed using a Vectastain ABC Kit (Vector Laboratories). The

specimens were then processed using the avidin-biotin complex method, and antibody

binding sites were visualized with 3,3’-diaminobenzidine tetrahydrochloride staining.

Surgically resected esophageal squamous cell carcinoma (ESCC) samples

(H2015-05284) were obtained from the patient. Formalin-fixed, paraffin-embedded

tissue sections were cut at a thickness of 4 μm. For heat-induced epitope retrieval,

deparaffinized sections in 0.01-mol/L citrate buffer were boiled 3 times for 5 minutes

using a microwave oven. The following antibodies were used for immunohistochemical

analysis of esophageal tissues: CD8 (1:3; Nichirei), CD68 (1: 1,600; Dako), and

HMGCR (1:100; Santa Cruz Biotechnology). For counting the number of The CD8-

positive or CD68-positive cells, four high-power fields (200×) were assessed in the

patient’s serial sections of muscle and ESCC tissues.

Immunoblotting. We compared HMGCR expression in the muscle tissue of the

presented case (MB15-35) with those of a healthy subject (P1234171, purchased from

BCH Research) and those of disease controls: MB15-15, MB15-23 and MB15-54.

Muscle and esophagus tissues were homogenized in CelLytic mammalian tissue

lysis/extraction reagent (Sigma-Aldrich). The total protein of 13 μg per sample were

separated by 5–20% SDS/PAGE and transferred to polyvinylidene difluoride

membranes. After blocking, the membranes were incubated with the mouse monoclonal

NEUROLOGY MS ID#: NEUROLOGY/2015/713974Tsujikawa et al.

anti-HMGCR antibody (1:1,000, Santa Cruz Biotechnology) at 4°C overnight. After

washing, the membranes were incubated with horseradish peroxidase-conjugated

secondary antibodies at a dilution of 1:5,000 at room temperature for 1 hour. The

proteins were detected with ECL Western blotting detection reagents (GE Healthcare).

An LAS-3000 imaging system (Fuji) was used to produce digital images. For

normalization, anti-GAPDH antibody (1:10,000; Abcam) was used as internal control of

protein loading.

e-Reference

e1) Muro Y, Sugiura K, Akiyama M. A new ELISA for dermatomyositis autoantibodies:

rapid introduction of autoantigen cDNA to recombinant assays for autoantibody

measurement. Clin Dev Immunol 2013;2013:856815.

NEUROLOGY MS ID#: NEUROLOGY/2015/713974Tsujikawa et al.

Figure e-1 Anti-HMGCR immunohistochemistry for ESCC tissues in serum anti-

HMGCR antibody-positive and -negative patients

The ESCC tissue of the presented case with anti-HMGCR antibody had more intensive

immunoreactivity to HMGCR protein than those of anti-HMGCR antibody-negative

patients, suggesting that the overexpression of HMGCR is possibly involved with the

production of serum anti-HMGCR antibodies. ESCC, esophageal squamous cell

carcinoma; y.o., years old; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A

reductase; and Ab, antibody. Bars: 40 μm

60 y.o. / Male 59 y.o. / Male72 y.o. / Male76 y.o. / Female

T2N3M0

Control cases

NegativeNegativeNegative PositiveAnti-HMGCR Ab

Age / Sex

TNM staging T1N3M0

ModerateModerateModerateDifferentiation Moderate

T1N3M0T3N2M0

H2015-02417H2015-14285H2014-04592 H2015-05284

Our case

NEUROLOGY MS ID#: NEUROLOGY/2015/713974Tsujikawa et al.

Figure e-2　 CD8-positive and CD68-positive cell count in muscle and tumor

The patient’s muscle and ESCC tissue had a similar relative proportion of CD8-positive

and CD68-positive cells. The average CD8/CD68 ratio was 0.4 ± 0.1 (mean ± S.D.) in

both the muscle and ESCC tissues, suggesting that the muscle and ESCC tissues share a

common inflammatory pathogenesis. ESCC, esophageal squamous cell carcinoma.

150

200

100

50

0

CD68CD8

Number of cells / field (200 ×)

ESCC

150

200

100

50

0

CD68

Muscle

CD8

Number of cells / field (200 ×)