ISSN 0030- 6096

O S A K A
C I T Y

Vol.

65

No.1

M E D I C A L

2019

J O U R N A L
V o l . 6 5 ,N o . 12 0 1 9PUBLISHED BYOSAKA CITY MEDICAL ASSOCIATIONOSAKA JAPANOsaka City Medical Journal
Vo l . 6 5 , N o . 1 ,June 2019

CONTENTS
page
Serum Myostatin Level in COPD Patients with and without Sarcopenia-Application for Biomarker of Sarcopenia DiagnosisSHINYA YOSHIDA, KAZUHISA ASAI, NAOKI IJIRI, YOHKOH KYOMOTO,TAMAKI KAWAMOTO, ATSUKO OKAMOTO, KAZUHIRO YAMADA,TETSUYA WATANABE, YOSHIHIRO TOCHINO, and TOMOYA KAWAGUCHI ････････････････････

1

Decreased Level of Phosphatase and Tensin Homolog Deleted from
Chromosome 10 (PTEN), a Negative Regulator of Phosphoinositide
3-kinase (PI3K)/Serine-Threonine Protein Kinase (Akt) Pathway,
is Related to Chronic Obstructive Pulmonary Disease
TAMAKI KAWAMOTO, KAZUHISA ASAI, KAZUHIRO YAMADA,SHINYA YOSHIDA, ATSUKO OKAMOTO, KANAKO SATO, NAOKI IJIRI,TETSUYA WATANABE, and TOMOYA KAWAGUCHI ･･････････････････････････････････････････････････

9

Focal Atrophy of the Dorsal Brainstem Correlates with Hallucinations inPatients with Dementia with Lewy Bodies
TADASHI OHTOMO, YASUNORI MATSUDA, KENTARO UCHIDA,JUMPEI MARUTA, and KOKI INOUE････････････････････････････････････････････････････････････････････ 19
Age-specific Differences in Insulin dose of Japanese Patients with Type 1
Diabetes of Various Ages
YUKO HOTTA, TOMOYUKI KAWAMURA, NAOKO NISHIKAWA,KAYAKO HASHIMURA, YONEO KASHIHARA, TOMOMI HASHIMOTO,
MASAKAZU HIROSE, TAKASHI HIGASHIDE, HARUO SHINTAKU,
and TAKASHI HAMAZAKI･･････････････････････････････････････････････････････････････････････････････････ 31
Clinical Aspects and Genetic Analysis of Pediatric Neurofibromatosis
Type 1 with Neurological Complications
KENJI FUJITA, KANAKO YAMASHITA, TAKAO HOSHINA,NORIKATSU HIKITA, TARO SHIMONO, KAZUYOSHI FUKAI,TOSHIKI TAKENOUCHI, TOMOKO UEHARA, KENJIRO KOSAKI,
HIDEYUKI SAYA, TAKASHI HAMAZAKI, and TOSHIYUKI SETO ･････････････････････････････････ 41
Change in the Thickness of Retinal Layers after Selective Retina
Therapy (SRT) in Patients with Central Serous Chorioretinopathy
KUMIKO HIRAYAMA, MANABU YAMAMOTO, TAKEYA KOHNO,DIRK THEISEN-KUNDE, RALF BRINKMANN, YOKO MIURA, and SHIGERU HONDA････････ 55
Assessment of Dual-time-point 18F-fluorodeoxyglucose-Positron Emission
Tomography/Computed Tomography Imaging of Malignant Tumors
Using Visually and Semiquantitatively: What Kind of Tumors is Useful?
HIROTAKA SEURA, TERUE OKAMURA, KOICHI KOYAMA,
and YUTAKA MASUOKA････････････････････････････････････････････････････････････････････････････････････ 65

Osaka City Med. J. Vol. 65, 1-8, 2019

Serum Myostatin Level in COPD Patients with and without
Sarcopenia -Application for Biomarker of Sarcopenia DiagnosisSHINYA YOSHIDA, KAZUHISA ASAI, NAOKI IJIRI, YOHKOH KYOMOTO, TAMAKI KAWAMOTO,ATSUKO OKAMOTO, KAZUHIRO YAMADA, TETSUYA WATANABE,YOSHIHIRO TOCHINO, and TOMOYA KAWAGUCHI
Department of Respiratory Medicine, Osaka City University Graduate School of Medicine

Abstract
Background
Chronic obstructive pulmonary disease (COPD) has significant extrapulmonary effects including
weight loss, nutritional abnormalities, and skeletal muscle dysfunction. Sarcopenia is one of several
comorbidities experienced by COPD patients, which may negatively affect quality of life and increasemortality. Previous studies have reported that exercise may reduce the severity of sarcopenia;
however, the mechanism is only partially understood. We hypothesized that levels of irisin andmyostatin, one of the myokines, are correlated with sarcopenia in COPD patients.
Methods
A total of 39 male COPD patients were enrolled, in addition to 30 male never-smokers as a control
group. Pulmonary function testing was performed on all participants, while body-composition
analysis was performed on the COPD patients to measure muscle mass and diagnose sarcopenia.
Serum irisin and myostatin levels were measured using a commercially available enzyme-linked
immunosorbent assay kit.
Results
The COPD patients were divided into a sarcopenia group (n＝9) and a non-sarcopenia group (n＝30). Serum myostatin levels were significantly lower in COPD patients with sarcopenia than in those
without sarcopenia, while there was no significant difference in irisin levels among COPD patientswith and without sarcopenia.
Conclusions
Serum myostatin level was correlated with muscle mass and is a potential biomarker forsarcopenia in COPD patients.
Key Words: Sarcopenia; Irisin; Myostatin; Myokine; COPD

Received August 30, 2018; accepted November 27, 2018.
Correspondence to: Kazuhisa Asai, MD.
Department of Respiratory Medicine, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-3916; Fax: +81-6-6646-6160
E-mail: kazuasai@msic.med.osaka-cu.ac.jp-1-

Yoshida et al

Introduction
Chronic obstructive pulmonary disease (COPD) is a common, preventable, and treatable disease
that is characterized by persistent respiratory symptoms and airflow limitation due to airway and/or alveolar abnormalities usually caused by significant exposure to noxious particles or gases. COPD
has significant extrapulmonary effects including weight loss, nutritional abnormalities, and skeletal
muscle dysfunction. Skeletal muscle dysfunction is characterized by both sarcopenia and abnormal
function of the remaining cells1).
As the human body ages, skeletal muscle mass declines annually by 0.1% to 0.5% starting from 30
years old, with a dramatic acceleration after 65 years old. This gradual decrease in muscle mass isaccompanied by a simultaneous reduction in strength. Excessive age-related loss of muscle mass andstrength is often referred to as primary sarcopenia 2). There is also a secondary sarcopenia related
to disease. Sarcopenia is among the comorbidities present in COPD patients and may negatively
impact quality of life. Therefore, early detection of sarcopenia can reduce the long-term care needsof this population, as well as improve quality of life and reduce morbidity and mortality3). Previous
studies have reported that exercise may reduce the severity of sarcopenia; however, the mechanism isonly partially understood.
Recently, myokines, such as irisin and myostatin, were discovered to be secreted from muscle
tissue. Irisin is secreted by proteolytic cleavage of the membrane protein fibronectin type Ⅲ domaincontaining protein 5 (FNDC5)4). Exercise triggers the cleavage of FNDC5 to secrete irisin into thebloodstream, which subsequently elevates energy expenditure in the subcutaneous adipose tissue
through adipocyte browning4). The beneficial role of irisin in skeletal muscle metabolism has been
described and it has been shown that irisin stimulates glucose uptake and lipid metabolism via
activation of AMP-activated protein kinase5,6).
On the other hand, myostatin, a member of the transforming growth factor-beta superfamily,
has been established to be a negative regulator of skeletal muscle mass and is mainly expressed inskeletal muscles7). Myostatin inhibits satellite cell proliferation and differentiation in an autocrine
and paracrine manner; conversely genetic deletion of myostatin leads to muscle hypertrophy inhumans and mice8). While myostatin activation negatively regulates muscle growth, myostatin
expression is downregulated after endurance and resistance exercise9). Myostatin is considered to be
negatively correlated with muscle mass because it is a negative regulator of muscle growth. Howevera recent study reported that plasma myostatin levels were positively correlated with lean body mass10).
The relationship between these myokines and muscle mass in COPD patients, however, has been
unclear. We hypothesized that myostatin and irisin levels are correlated with sarcopenia in COPD
patients. To address this hypothesis, we measured serum myostatin and irisin levels, and evaluatedthe relationships between these myokines and sarcopenia and muscle mass.

Methods
Subjects
Thirty-nine male COPD patients were recruited prospectively in the outpatient departmentof Osaka City University Hospital (Osaka, Japan) between April 2016 and December 2016. All
COPD patients were stable outpatients and diagnosed with COPD according to the Global Initiative
for Chronic Obstructive Lung Disease guidelines11). They had no history of malignant diseases,
pulmonary comorbidities and oral corticosteroid therapy. We recruited male never-smokers older-2-Myostatin in COPD Patients

than 70 years as controls from a group of healthy volunteers at Medcity21, medical examinations
clinics between March 2016 and October 2017.
This study was approved by the Ethics Committee of Osaka City University Hospital (approval
No. 3330), and all patients provided informed written consent for participation. All procedures were
performed according to the guidelines of the Declaration of Helsinki.
Body composition analysis (BIA)
Body-composition analysis was performed in the COPD patients to measure BMI, muscle mass
(MM), fat-free mass (FFM), FFM index (FFMI), and skeletal muscle mass index (SMI), using
bioelectrical impedance analysis using the InBody 3.0 system analyzer (InBody, Seoul, South Korea).
We didn t check their ingestion of food or drink before BIA. SMI is defined as appendicular skeletal
muscle mass (ASM) divided by the square of height. ASM is calculated by summing the muscle massof the four limbs. Sarcopenia was defined according to the Asian Working Group for Sarcopenia(AWGS) standard, with SMI ＜7 kg/m2 and grip strength ＜26 kg.
Myokine measurements
For each subject, peripheral venous blood was drawn at the rest and serum samples were deep
frozen and kept at −80℃ until the measurement. Serum irisin levels were measured using acommercially available enzyme-linked immunosorbent assay (ELISA) kit (Phoenix Pharmaceuticals,
Burlingame, CA, USA) according to the manufacturer s protocol. Serum myostatin levels also were
measured using an ELISA kit (R&D systems Inc., Minneapolis, USA) according to the manufacturer s
protocol.
Statistical analysis
Data are expressed as mean ±standard deviation unless otherwise indicated. Baseline differenceswere determined using Student s t test or the Mann-Whitney U test. Group of data that failed tests
for normality and equal variance were analyzed by the nonparametric Kruskal-Wallis analysis ofvariance followed by Dunn s test. Associations between continuous variables were described by
Spearman s correlation coefficients for variables that were not normally distributed. Statisticalanalyses were performed using JMP version 10.0.0 (SAS Institute, Cary, NC, USA) for Windows(Microsoft Corporation, Redmond, WA, USA). In all statistical analyses, p＜0.05 was considered to bestatistically significant.

Results
Patient characteristics
The characteristics of the 39 COPD patients and 30 healthy controls are summarized in Table 1.
There were no significant differences in age or BMI between the COPD patients and healthy controls.
As shown in Table 1, there was no significant difference in FVC and %FVC between the two groups.
However, FEV1 was significantly lower in COPD patients than in healthy controls (1.8±0.6 vs 2.7±0.4,
respectively; p＜0.0001) and FEV1 (% predicted) was also significantly lower in COPD patients thanin healthy controls (66.4±21.1 vs 97.3±14.6, respectively; p＜0.0001).
COPD patients were divided in two groups, the sarcopenia group (n＝9) or the non-sarcopenia (n＝30) group in accordance with AWGS standards (Table 2). There was no significant difference in FVC,
FEV1, FEV1/FVC or BMI between COPD patients with and without sarcopenia. However, MM, FFMI,
and SMI were significantly lower in COPD patients with sarcopenia than in those without sarcopenia(p＜0.0005).

-3-

Yoshida et al
Table 1. Characteristics of all participants
COPD

healthy controls

p value

Subject No.

39

30

Age (years)

74±8

74±3

NS

BMI (kg/m2)

23.3±3.6

23.0±2.9

NS

FVC (L)

3.3±0.8

3.4±0.5

NS%FVC (%)

100.2±16.9

98.8±13.7

NS

FEV1 (L)

1.8±0.6

2.7±0.4＜0.0001%FEV1 (%)

66.4±21.1

97.3±14.6＜0.0001

FEV1/FVC (%)

51.9±13.978.8±3.9＜0.0001

COPD, chronic obstructive pulmonary disease; BMI, body mass index; FVC, forced vital capacity; andFEV1, forced expiratory volume in 1 second.

Table 2. Characteristics of COPD patients with and without sarcopenia
non-sarcopenia

sarcopenia

p value

Subject No.

30

9GOLD stage Ⅰ, n

91GOLD stage Ⅱ, n

12

7GOLD stage Ⅲ, n

8

1

GOLD stage Ⅳ, n

10

FVC (L)

3.5±0.9

2.9±0.3

NS%FVC (%)

101±1897±10NSFEV1 (L)

1.8±0.7

1.6±0.6

NS

%FEV1

66±20

67±23

NS

FEV1/FVC (%)

51±14

53±14

NS

Age (yr)

73±8

80±5

＜0.05

BMI (Kg/m2)

23.8±3.2

21.7±3.6NSMM

46.5±6.2

37.1±3.5＜0.001FFMI

17.8±1.515.6±1.5＜0.0005

SMI (Kg/m2)

7.3±0.8

6.1±0.6＜0.0005

COPD, chronic obstructive pulmonary disease; GOLD, Global Initiative for Chronic Obstructive Lung
Disease; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 second; BMI, body mass index; MM,muscle mass; FFMI, fat free mass index; and SMI, skeletal muscle mass index.

Relationships between myokine levels and body composition
In COPD patients, serum irisin levels were positively correlated with FFMI (r＝0.36, p＜0.05) andSMI (r＝0.40, p＜0.01), but not significantly correlated with MM (r＝0.32, p＞0.05) (Fig. 1). However,serum myostatin levels were positively correlated with all body composition indices including MM(r＝0.44, p＜0.005), FFMI (r＝0.50, p＜0.01), and SMI (r＝0.54, p＜0.005) (Fig. 2).

-4-

Myostatin in COPD Patients

Figure 1. Correlations of serum irisin levels with MM, FFMI, and SMI. A, There was no significant correlation
between serum irisin levels and MM. B and C, There were significant positive correlations between serum irisin levels
and FFMI and SMI. MM, muscle mass; FFMI, fat free mass index; and SMI, skeletal muscle mass index.

Figure 2. Correlations of serum myostatin levels with MM, FFMI, and SMI. A-C, There were significant positive
correlations between serum myostatin levels and MM, FFMI, and SMI. MM, muscle mass; FFMI, fat free mass index;
and SMI, skeletal muscle mass index.

Figure 3. Comparison of serum myostatin levels
between healthy controls and COPD patients with andwithout sarcopenia. There was a significant difference
in serum myostatin levels between healthy controls
and COPD patients with sarcopenia. COPD, chronic
obstructive pulmonary disease.

Figure 4. Comparison of serum irisin levels between
COPD patients with and without sarcopenia. There wasno significant difference in serum irisin levels between
COPD with and without sarcopenia. COPD, chronic
obstructive pulmonary disease.

-5-

Yoshida et al

Sarcopenia and myostatin levels
Serum myostatin levels in healthy controls were significantly higher than COPD patients withsarcopenia (4.01±0.25 ng/mL vs 2.76±0.20 ng/mL; p＜0.05) but there was no significant difference
in serum myostatin levels between healthy controls and those without sarcopenia. Serum myostatinlevels were significantly lower in COPD patients with sarcopenia than in those without sarcopenia(2.76±0.20 ng/mL vs 3.53±0.19 ng/mL; p＜0.05) (Fig. 3). There was no significant difference in serumirisin levels between COPD patients with sarcopenia and those without sarcopenia (3.94±0.68 ng/mL
vs 5.10±0.33 ng/mL; p＞0.1) (Fig. 4).

Discussion
In this study, we examined body composition and sarcopenia in COPD patients, and revealed thatirisin and myostatin, one of the myokines, were positively correlated with muscle mass parameters
including FFMI and SMI. Sarcopenia is a comorbidity in COPD, and is characterized by the loss ofskeletal muscle mass and strength, which is associated with low exercise capacity, lower quality oflife, and increased mortality12,13).
The assessment of body composition measured using bioelectrical impedance analysis (BIA) or
dual energy X-ray absorptiometry (DXA) are among the diagnostic methods for sarcopenia. BIA
measures the resistance to flow of an electrical current as it passes through the body and is reflectiveof body composition. DXA calculates body density and body composition by irradiating the body with
two X-ray beams. Early detection of sarcopenia in COPD patients is important. However, in many
clinics and hospitals, body composition cannot be measured due to a lack of these devices; thus early
detection of sarcopenia is difficult. Alternatively, we focused on myokine levels, such as irisin andmyostatin, to be potential biomarkers of sarcopenia.
Myostatin is mainly produced by skeletal muscles and served as a negative regulator of muscle
growth and it is released into plasma in a form of precursor protein, which can be cleaved into mature
myostatin by bone morphogenetic-1/tolloid family of metalloproteases. Some studies have reported
that postnatal inhibition of myostatin unequivocally increased skeletal muscle mass in adults andolder mammals14,15). Another study reported that weekly injection of a neutralizing antibody tomyostatin for 4 weeks significantly increased the relative weights of individual muscles by up to
17% in aged mice and improved indices of muscle performance and whole-body metabolism16). The
other study reported that blocking myostatin might be a therapeutic target to combat sarcopenia
and inhibiting myostatin-activin A signaling is therefore a highly promising therapeutic strategy tocombat muscle loss in older people17). Therefore, myostatin was considered to be a negative regulatorof muscle growth.
However, a recent study demonstrated a positive correlation between plasma myostatin levels
and lean body mass11). Our study also revealed a positive correlation between serum myostatin levels
and muscle mass. We speculate that hypertrophied muscle may secrete more myostatin as negative
feedback to inhibit excessive muscle hypertrophy. Our study demonstrated that myostatin levels
were significantly lower in COPD patients with sarcopenia than in those without sarcopenia. This
result suggests that myostatin may be a potential biomarker for sarcopenia in COPD patients.
Previous studies have also reported that serum C1q, C-terminal agrin fragment, and p53 codon 72
polymorphism may be also be potential biomarkers of sarcopenia18-20). On the other hand, there wasno significant difference in serum irisin levels between COPD patients with and without sarcopenia.

-6-

Myostatin in COPD Patients

Considering the different results between myostatin and irisin, we speculated that myostatinwould be mainly correlated with muscle mass but irisin would be correlated with muscle massand other metabolic disorder such as diabetes mellitus. As a result, serum irisin levels might beinfluenced by not only muscle mass but also other metabolic factors.
In conclusion, myostatin is a potential biomarker of sarcopenia in COPD patients. However, there
were some limitations to this study, among which include its single-center, cross-sectional designand limited sample size; therefore, causal relationships could not be determined. Second, this study
enrolled only male COPD patients; therefore, it also remains unknown whether these results apply
to female COPD patients. Third, twenty-four out of thirty-nine COPD patients overlapped withthose in the previous report21) which our group reported. Fourth, we didn t estimate the difference inamount of muscle mass between COPD patient and healthy controls because we didn t measure bodycomposition in healthy controls.
Further studies are needed to better understand the pathophysiology of sarcopenia in COPD andto determine other potential biomarkers.
Acknowledgments
All authors have no COI to declare regarding the present study.
We acknowledge Tatsuo Kimura and Shinya Fukumoto (Medcity 21, medical examinations clinics)
for providing serum samples and clinical data of never-smokers enrolled in this study.
References
1. Munhoz da Rocha Lemos Costa T, Costa FM, Jonasson TH, et al. Body composition and sarcopenia in patientswith chronic obstructive pulmonary disease. Endocrine 2018;60:95-102.
2. Liguori I, Russo G, Aran L, et al. Sarcopenia: assessment of disease burden and strategies to improveoutcomes. Clin Interv Aging 2018;13:913-927.
3. Chang JS, Kim TH, Nguyen TT, et al. Circulating irisin levels as a predictive biomarker for sarcopenia: across-sectional community-based study. Geriatr Gerontol Int 2017;17:2266-2273.
4. Boström P, Wu J, Jedrychowski MP, et al. A PGC1-α-dependent myokine that drives brown-fat-likedevelopment of white fat and thermogenesis. Nature 2012;481:463-468.
5. Huh JY, Dincer F, Mesfum E, et al. Irisin stimulates muscle growth-related genes and regulates adipocyte
differentiation and metabolism in humans. Int J Obes (Lond) 2014;38:1538-1544.
6. Lee HJ, Lee JO, Kim N, et al. Irisin, a novel myokine, regulates glucose uptake in skeletal muscle cells via
AMPK. Mol Endocrinol 2015;29:873-881.
7. McNally EM. Powerful genes--myostatin regulation of human muscle mass. N Engl J Med 2004;350:26422644.
8. Huh JY. The role of exercise-induced myokines in regulating metabolism. Arch Pharm Res 2018;41:14-29.
9. Allen DL, Hittel DS, McPherron AC. Expression and function of myostatin in obesity, diabetes, and exercise
adaptation. Med Sci Sports Exerc 2011;43:1828-1835.
10. Wu LF, Zhu DC, Wang BH, et al. Relative abundance of mature myostatin rather than total myostatin isnegatively associated with bone mineral density in Chinese. J Cell Mol Med 2018;22:1329-1336.
11. Vogelmeier CF, Criner GJ, Martinez FJ, et al. Global strategy for the diagnosis, management, and preventionof chronic obstructive lung disease 2017 report. GOLD executive summary. Am J Respir Crit Care Med
2017;195:557-582.
12. Tunsupon P, Lal A, Abo Khamis M, et al. Comorbidities in patients with chronic obstructive pulmonary
disease and pulmonary rehabilitation outcomes. J Cardiopulm Rehabil Prev 2017;37:283-289.
13. Polkey MI, Moxham J. Attacking the disease spiral in chronic obstructive pulmonary disease. Clin Med (Lond)
2006;6:190-196.
14. Whittemore LA, Song K, Li X, et al. Inhibition of myostatin in adult mice increases skeletal muscle mass andstrength. Biochem Biophys Res Commun 2003;300:965-971.
15. LeBrasseur NK, Schelhorn TM, Bernardo BL, et al. Myostatin inhibition enhances the effects of exercise on-7-

Yoshida et al
performance and metabolic outcomes in aged mice. J Gerontol A Biol Sci Med Sci 2009;64:940-948.
16. White TA, LeBrasseur NK. Myostatin and sarcopenia: opportunities and challenges - a mini-review.
Gerontology 2014;60:289-293.
17. Cohen S, Nathan JA, Goldberg AL. Muscle wasting in disease: molecular mechanisms and promisingtherapies. Nat Rev Drug Discov 2015;14:58-74.
18. Watanabe S, Sato K, Hasegawa N, et al. Serum C1q as a novel biomarker of sarcopenia in older adults.
FASEB J 2015;29:1003-1010.
19. Drey M, Sieber CC, Bauer JM, et al. C-terminal Agrin Fragment as a potential marker for sarcopenia caused
by degeneration of the neuromuscular junction. Exp Gerontol 2013;48:76-80.
20. Di Renzo L, Gratteri S, Sarlo F, et al. Individually tailored screening of susceptibility to sarcopenia using p53
codon 72 polymorphism, phenotypes, and conventional risk factors. Dis Markers 2014;2014:743634.
21. Sugiyama Y, Asai K, Yamada K, et al. Decreased levels of irisin, a skeletal muscle cell-derived myokine, arerelated to emphysema associated with chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon
Dis 2017;12:765-772.

-8-

Osaka City Med. J. Vol. 65, 9-17, 2019

Decreased Level of Phosphatase and Tensin Homolog Deleted from
Chromosome 10 (PTEN), a Negative Regulator of Phosphoinositide
3-kinase (PI3K)/Serine-threonine Protein Kinase (Akt) Pathway,
is Related to Chronic Obstructive Pulmonary Disease
TAMAKI KAWAMOTO, KAZUHISA ASAI, KAZUHIRO YAMADA, SHINYA YOSHIDA, ATSUKO OKAMOTO,KANAKO SATO, NAOKI IJIRI, TETSUYA WATANABE, and TOMOYA KAWAGUCHI
Department of Respiratory Medicine, Osaka City University Graduate School of Medicine

Abstract
Background
Cigarette smoking causes persistent airway inflammation and is considered to be the major causeof chronic obstructive pulmonary disease (COPD). The activation of phosphoinositide 3-kinase(PI3K)/serine-threonine protein kinase (Akt) pathway is known to have an important role in COPD
pathogenesis. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is a negative
regulator of the PI3K/Akt pathway and is the gene most significantly associated with COPD.
However, the role of PTEN in COPD pathogenesis remains unclear.
Methods
Bronchial epithelial cells were obtained by bronchial brushing technique from patients with
COPD, healthy smokers, and healthy non-smokers. PTEN and PI3Kδ expression in bronchial
epithelial cells were examined by real-time polymerase chain reaction (PCR) and western blotting.
Moreover, we investigated the correlation of PTEN and PI3KCD (encoding PI3Kδ) with pulmonary
function and clinical parameters.
Results
Forty-nine COPD patients, 31 healthy smokers, and 22 healthy non-smokers were enrolled in thisstudy. In COPD patients, PTEN expression was significantly decreased in bronchial epithelial cells
and negatively correlated with pack-year (r ＝−0.40, p ＜0.001). PTEN mRNA expression was
significantly correlated with the parameters of obstructive impairment, such as %FEV1 (r＝0.26 p＜0.01), FEV1/FVC, (r＝0.20, p＝0.04) and %diffusing capacity of the lung for carbon monoxide (DLco)(r＝0.34, p＜0.01). There was no significant difference in PI3Kδ mRNA levels between each group.
Conclusions
PTEN expression in COPD patients was significantly lower than that in healthy non-smokers,

Received August 30, 2018; accepted November 27, 2018.
Correspondence to: Kazuhisa Asai, MD, PhD.
Department of Respiratory Medicine, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan
Tel: +81-6-6645-3821; Fax: +81-6-6636-0439
E-mail: kazuasai@med.osaka-cu.ac.jp-9-Kawamoto et alsuggesting that decrease of PTEN expression might be related to pathogenesis of COPD.
Furthermore, the decrease in PTEN expression was correlated with obstructive impairment of COPD.
PTEN might be a possible therapeutic target for COPD.
Key Words: ‌Chronic obstructive pulmonary disease (COPD); phosphatase and tensin
homolog deleted from chromosome 10 (PTEN); phosphoinositide 3-kinase(PI3K)/serine-threonine protein kinase (Akt) pathway

Introduction
Chronic obstructive pulmonary disease (COPD) is a global health problem. The World HealthOrganization estimated that more than 3 million people died from COPD in 2005 which corresponds
to 5% of all deaths globally, and COPD will be the third leading cause of death by 20301). COPD ischaracterized by progressive irreversible airflow limitation and chronic inflammation of the airways2).
Cigarette smoke is the major risk factor of COPD3-5). Oxidative stress caused by cigarette smoke
damages lipids, proteins, and DNA and induces inflammatory reactions in the lung6-8). Even if
cigarette smoke exposure is stopped, the disease may still progress due to ageing and persistence of
inflammation9,10). In vitro studies have shown that oxidative stress by hydrogen peroxide (H2O2)
activates phosphoinositide 3-kinase (PI3K) and serine/threonine protein kinase (Akt) 11,12). A recent
study suggested that persistent activation of Akt is implicated in the pathogenesis of COPD13). Akt is
activated by phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 is converted to phosphatidylinositol-4,5bisphosphate (PIP2) through PI3K, which is encoded by the PI3Kδ gene in humans.
Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) was originally isolated asa tumor suppressor gene and is known to be a negative regulator of PI3K. PTEN converts PIP3 backto PIP2, antagonizing PI3K action; thus, the activation of PTEN regulates Akt signaling pathway
negatively14-16). PTEN is reported to be the most significantly associated gene with COPD17). Moreover,previous study has demonstrated that the protein expression levels of PTEN are significantly
decreased in the lung tissue of emphysematous mice exposed to cigarette smoke18). Another study has
shown that PTEN protein is decreased in lung homogenate of COPD patients 19). PTEN may berelated to the pathogenesis of COPD, however, in humans, there are few reports that reveal the levelsof PTEN in COPD patients. A recent study on human bronchial biopsies of COPD patients has
demonstrated an increased inflammatory gene and protein expression and structural alterations in
bronchial epithelial cells, which suggests that these cells may be the key effector in the pathogenesis
of COPD20).
Therefore, we hypothesized that impaired expression of PTEN in human bronchial epithelial cells
would be a possible cause of COPD. To address this hypothesis, PTEN and PI3Kδ expression inhuman bronchial epithelial cells were measured by real-time polymerase chain reaction (PCR) andwestern blotting. Moreover, we examined the correlation of PTEN and PI3Kδ with pulmonary
functions and clinical parameters.

Methods
Subjects
COPD patients, healthy smokers, and healthy non-smokers were enrolled in this study. COPD
was diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD)
guidelines21). Healthy smokers were current or former smokers with more than 10 pack-year history- 10 -

Impaired PTEN Expression in COPD

who had no clinically significant medical disorders. All healthy non-smokers were never smokers
who had no clinically significant medical disorders. These subjects had no acute exacerbation andpneumonia for at least 2 weeks. All the subjects underwent pulmonary function test by spirometry(Chestac-25F, Chest Co., Tokyo, Japan). The diffusing capacity of the lung for carbon monoxide(DLco) was measured at least twice by the single-breath method. Bronchoscopy was performed in all
subjects to diagnose small nodules or the examination of bloody sputum at Osaka City University.
During bronchoscopic procedure, bronchial epithelial cells were obtained from the normal side bybronchial brushing. This study was approved by the ethics committee of Osaka City University
Hospital (approval number 1819), and all subjects gave written informed consent for their
participation.
Real-time PCR
Total RNA was extracted from bronchial epithelial cells using TRIzol (Life Technologies Japan,
Tokyo, Japan). After quantification, 1 μg of RNA was reverse transcribed into complementary DNA
with a SuperScript VILO cDNA Synthesis Kit® (Thermo Fisher Scientific, Carlsbad, CA, USA).
Quantitative real-time PCR was performed using an Applied Biosystems 7500 Real-Time PCR System(Life Technologies Japan, Tokyo, Japan) with TaqMan Gene Expression Assays (Thermo Fisher
Scientific) for PTEN (Hs02621230_s1), PI3Kδ (Hs00192399_m1) and glycer-aldehyde 3-phosphate
dehydrogenase (GAPDH) (Hs99999905_m1).
Western blot analysis
Brushed bronchial epithelial cells were suspended in 4 mL of phosphate buffered saline (PBS) andcentrifuged at 1500×g at 4℃ for 10 minutes. Supernatant was discarded, and pellet was dissolved in
100 μL of cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). BCA Protein Assay Kit(Pierce, Waltham, MA, USA) was used for protein quantification. Proteins for each sample wereseparated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto
polyvinylidene difluoride (PVDF) membrane by western blotting using Mini-PROTEAN® TGX™
Precast Protein Gels (4561023, Bio-Rad, Hercules, California, USA). Membranes were incubatedwith anti-PTEN (accession number: P60484) rabbit IgG antibody (ab32199, Abcam, 1:2000 dilution)or anti-GAPDH (accession number: P04406) antibody (ab128915, Abcam, 1:10000 dilution) overnightat 4℃, and further incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG
secondary antibody (sc-2004, Santa Cruz Biotechnology) for 2 hours at room temperature. The
densitometry analysis was performed to measure protein level in PTEN and GAPDH using LAS4000(Fuji Imaging, Tokyo, Japan).
Statistical analysis
GraphPad Prism version 7.04 (GraphPad Software, San Diego, CA, USA) was used for Statisticalanalysis. To compare two groups, Student's t-test and for more than three groups, one-way analysis
of variance (ANOVA) with Tukey-Kramer post hoc test was performed. The significance of correlation
was analyzed by Spearman's rank correlation coefficients. In all statistical analyses, p-values ＜0.05
were considered significant.

Results
Subject characteristics
Forty-nine COPD patients, 31 healthy smokers, and 22 healthy non-smokers were enrolled in thisstudy. The characteristics of the subjects and parameters of pulmonary function test are shown in- 11 -

Kawamoto et al

Table 1. COPD patients were in GOLD stage Ⅰ-Ⅳ (25/16/7/1). There was a significant difference interm of sex ratio between the three groups, for instance, in case of COPD, the proportion of males washigher than females. However, in terms of age and body mass index (BMI) there was no significant
difference between the three groups. %FEV1 values were significantly lower in COPD patientscompared to healthy non-smokers (p＜0.001).
PTEN and PI3Kδ expression in COPD patients
PTEN mRNA levels were significantly decreased in COPD patients (p ＜0.001) and healthy
smokers (p＝0.004) compared to healthy non-smokers (Fig. 1A). On the other hand, there was no
significant difference in PI3Kδ mRNA level between the three groups (Fig. 1B). Moreover, PTEN
protein level in COPD patients was significantly lower than that of healthy non-smokers (p＝0.013)(Figs. 2A and 2B).

Table 1. Subjects characteristics
Healthy non-smoker

Healthy smoker

COPD

Subjects No.

22

31

49

Sex (male/female)

6/16

26/5

42/7

p＜0.001＊

67.1 (62.6-71.5)

67.6 (64.0-71.2)

69.4 (67.7-71.2)

N.S.

2 ＊

22.1 (20.8-23.5)

21.5 (20.1-23.0)

20.9 (19.9-22.0)

N.S.

Age (year)

BMI (kg/m )Smoking history(Pack-Year)

FEV1 (% predicted)
＊

0

＊

＊

130.7 (120-142)

48.8

＊＊

(38.5-59.1)

122 (112-132)

60.4
86

＊＊

＊＊

p-value(52.8-68.0)

p＜0.001(76.3-96.3)

p＜0.001＊＊:mean (95% confidence interval):p＜0.05 compared with healthy non-smoker.
COPD, chronic obstructive pulmonary disease; BMI, body mass index; and FEV1, forced expiratory volume in one
second.

Figure 1. PTEN and PI3Kδ mRNA expression in study subjects. A, PTEN mRNA levels were significantly decreased
in COPD patients and healthy smokers compared to healthy non-smokers. Each bar represents the mean value.
B, There was no significant difference in PI3Kδ mRNA level between the three groups. Each bar represents themean value. COPD, chronic obstructive pulmonary disease; PTEN, phosphatase and tensin homolog deleted fromchromosome 10; PI3K, phosphoinositide 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; and N.S., notsignificant.

- 12 -

Impaired PTEN Expression in COPD

Figure 2. A and B, Western blotting analysis of PTEN in COPD patients and healthy non-smokers. PTEN protein
level in COPD patients (n＝4) was significantly lower (0.2±0.3) than that of healthy non-smokers (n＝4) (1.9±0.9).
Statistical analyses were performed using the Student's t-test. Values are means±standard deviation. COPD, chronic
obstructive pulmonary disease; PTEN, phosphatase and tensin homolog deleted from chromosome 10; and GAPDH,
glycer¬aldehyde 3-phosphate dehydrogenase.

Correlation between PTEN mRNA level and clinical parameters
There was a negative correlation between PTEN mRNA levels and pack-year (Fig. 3; r＝−0.40, p＜
0.001). However, there was no correlation of PTEN mRNA levels with age and BMI (data not shown).
There were significant positive correlations between PTEN mRNA levels and %FEV1 (r＝0.26, p＜0.01), FEV1/FVC (r＝0.20, p＝0.04), and %DLco (r＝0.34, p＜0.01) (Figs. 4A-4C). In contrast, PI3Kδ
mRNA levels were not significantly correlated with any parameters of pulmonary function test (Figs.
5A-5C).

Figure 3. Relationship between PTEN mRNA expression and pack year. There was a significant correlation between
PTEN mRNA levels and pack year. PTEN, phosphatase and tensin homolog deleted from chromosome 10; andGAPDH, glyceraldehyde 3-phosphate dehydrogenase.

- 13 -

Kawamoto et al

Figure 4. Correlations of PTEN mRNA levels with pulmonary function test values. A: %FEV1, B: FEV1/FVC, and
C: %DLco. There were significant positive correlations between PTEN mRNA levels and %FEV1, FEV1/FVC, and%DLco. PTEN, phosphatase and tensin homolog deleted from chromosome 10; GAPDH, glyceraldehyde 3-phosphate
dehydrogenase; FEV1, forced expiratory volume in one second; FVC, forced vital capacity; and DLco, diffusing capacityof carbon monoxide.

Figure 5. Correlations of PI3Kδ mRNA levels with pulmonary function test values. A: %FEV1, B: FEV1/FVC, and C:%DLco. No significant correlations were observed between PI3Kδ mRNA levels and %FEV1, FEV1/FVC, and %DLco.
PI3K, phosphoinositide 3-kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FEV1, forced expiratory volume
in one second; FVC, forced vital capacity; DLco, diffusing capacity of carbon monoxide; and N.S., not significant.

- 14 -

Impaired PTEN Expression in COPD

Discussion
In the present study, we found that PTEN expression was significantly decreased in humanbronchial epithelial cells of COPD patients, and it was related to pack-year and pulmonary functions.
Previous studies have reported that most inflammatory cells related to COPD were controlled byclass I PI3Ks22,23). There are four class Ⅰ PI3K isoforms in mammals (p110α, p110β, p110γ, and p110δ),
named after their respective p110 catalytic subunits. Among these isoforms, PI3Kδ (p110δ) distinctly
correlates with inflammation 24,25). Additionally, we examined the mRNA level of PI3Kδ in the
bronchial epithelial cells, however, we found no significant difference between groups. A recent study
indicates that persistent activation of Akt is strongly implicated in the etiology of COPD11). PI3K/Akt
pathway is mainly regulated by PI3K and PTEN. PI3K phosphorylates PIP2 to PIP3 and activates
the PI3K/Akt signaling pathway. In contrast, PTEN negatively regulates PI3K signaling by
dephosphorylating PIP3 to PIP215,16). The failure of these regulation mechanisms might be involved in
the progression of pathogenesis of COPD.
In an emphysematous mouse model, it was observed that there was notably increased
phosphorylated-Akt (p-Akt) expression and decreased PTEN expression in the lung tissue 18). In
humans, it has been reported that PTEN was the most significantly associated gene with COPD, and
two PTEN single nucleotide polymorphism (SNP)s (rs701848, rs1903858) were observed to be
associated with COPD risk among heavy smokers 17). To our knowledge, there are few reports
regarding regulation of PI3K/Akt signaling by PTEN in COPD patients. Our results indicated that in
COPD patients, persistent activation of Akt signaling pathway is caused by decreased PTEN
expression rather than increased PI3Kδ expression. However, the mechanism underlying thedecreased expression of PTEN in COPD is still unknown.
We presumed that smoking might be related to PTEN expression, and thus, examined therelationship between PTEN mRNA levels and pack-year. We confirmed that there was a negative
correlation between PTEN mRNA levels and pack-year. Although we had considered the possibility
that the decrease in PTEN expression was possibly affected by aging, there was no correlation
between PTEN mRNA levels and age in our study. Decreased expression of PTEN was reported inlung homogenates of COPD patients. PTEN expression was decreased following cigarette smoke
extract (CSE) stimulation in BEAS-2B cells19). Recent studies have reported that oxidative stressdecreases PTEN expression 26), and Nuclear factor erythroid 2-related factor 2 (Nrf2) is the main
regulator of antioxidant response27). We previously reported that Nrf2 expression was significantly
decreased in bronchial epithelial cells in COPD patients, and Nrf2 had a protective role against CSEinduced apoptosis28). Cigarette smoke contains reactive oxygen species (ROS) that can cause oxidative
stress29). This oxidative stress might have decreased PTEN expression in COPD patients.
We examined the correlation of PTEN and PI3Kδ with pulmonary functions. We found significant
positive correlations between PTEN mRNA levels and the parameters of obstructive impairment,such as %FEV1, FEV1/FVC, and %DLco. Yanagisawa et al reported that PTEN protein levels weresignificantly decreased in the peripheral lung of COPD patients. There was a significant positive
correlation between the PTEN protein levels and FEV1/FVC or %FEV1, indicating that the PTEN
levels were decreased significantly as COPD progresses19). These results were consistent with our
results.
In COPD, neutrophils play an important role in chronic inflammation30). The number ofneutrophils, the level of IL-6 and IL-8 in sputum or broncho-alveolar lavage fluid of COPD patients- 15 -

Kawamoto et al

were reported to be negatively correlated with severities of air flow limitation31-35). Akt activation has
been implicated in neutrophil migration and polarization36,37). PTEN has been shown to control
inflammation by suppressing PI3K/Akt pathway38). These results suggest that PTEN is related to thepathogenesis of COPD through chronic neutrophilic inflammation. PTEN may be a possible
therapeutic target for COPD.
Our study has some limitations. First, the study sample size was small. Second, we acquired
bronchial epithelial cells from approximately the 4th branch, not the alveolar region because oftechnical reasons. Third, there was a significant difference in the sex ratio between each group.
Since smokers and COPD patients were mostly men, it was difficult to align the sex ratio. However,to the best of our knowledge, there was no report that PTEN expression was affected by gender.
Despite these limitations, we could evaluate the level of PTEN in COPD patients and provide a firststep to understand the etiology of COPD.
In conclusion, in COPD patients, PTEN expression was significantly decreased in bronchial
epithelial cells. Furthermore, we found significant positive correlations between PTEN mRNA levels
and the parameters of obstructive impairment. Our study suggests that the decrease of PTEN
expression might be related to pathogenesis of COPD and PTEN might be a possible therapeutictarget for COPD. Further research is needed to confirm our findings.
Acknowledgments
All authors have no COI to declare regarding the present study.
This work was supported by grants from the Japan Society for the Promotion of Science (JSPS)
Kakenhi grant 15K09185.
References
1. World Health Organization. Burden of COPD. Available from:http://www.who.int/respiratory/copd/burden/en/
2. Vogelmeier CF, Criner GJ, Martinez FJ, et al. Global Strategy for the Diagnosis, Management, and Preventionof Chronic Obstructive Lung Disease 2017 Report: GOLD Executive Summary. Arch Bronconeumol 2017;53:128-149.
3. Hogg JC, Timens W. The pathology of chronic obstructive pulmonary disease. Annu Rev Pathol 2009;4:435459.
4. The Surgeon General's 1989 Report on Reducing the Health Consequences of Smoking: 25 Years of Progress.
MMWR Suppl 1989;38:1-32.
5. Lapperre TS, Postma DS, Gosman MM, et al. Relation between duration of smoking cessation and bronchialinflammation in COPD. Thorax 2006;61:115-121.
6. Zinellu E, Zinellu A, Fois AG, et al. Circulating biomarkers of oxidative stress in chronic obstructive
pulmonary disease: a systematic review. Respir Res 2016;17:150.
7. Rahman I. The role of oxidative stress in the pathogenesis of COPD: implications for therapy. Treat Respir
Med 2005;4:175-200.
8. van Eeden SF, Sin DD. Oxidative stress in chronic obstructive pulmonary disease: a lung and systemic
process. Can Respir J 2013;20:27-29.
9. Donohue JF. Ageing, smoking and oxidative stress. Thorax 2006;61:461-462.
10. Rutgers SR, Postma DS, ten Hacken NH, et al. Ongoing airway inflammation in patients with COPD who do
not currently smoke. Thorax 2000;55:12-18.
11. To Y, Ito K, Kizawa Y, et al. Targeting phosphoinositide-3-kinase-delta with theophylline reverses
corticosteroid insensitivity in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2010;
182:897-904.
12. Mercado N, Ito K, Barnes PJ. Accelerated ageing of the lung in COPD: new concepts. Thorax 2015;70:482-489.
13. Bozinovski S, Vlahos R, Hansen M,et al. Akt in the pathogenesis of COPD. Int J Chron Obstruct Pulmon Dis
2006;1:31-38.

- 16 -

Impaired PTEN Expression in COPD
14. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 2007;129:1261-1274.
15. Stambolic V, Suzuki A, de la Pompa JL, et al. Negative regulation of PKB/Akt-dependent cell survival by thetumor suppressor PTEN. Cell 1998;95:29-39.
16. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger,
phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998;273:13375-13378.
17. Hosgood HD 3rd, Menashe I, He X, et al. PTEN identified as important risk factor of chronic obstructive
pulmonary disease. Respir Med 2009;103:1866-1870.
18. Lu J, Xie L, Liu C, et al. PTEN/PI3k/AKT regulates macrophage polarization in emphysematous mice. Scand
J Immunol 2017;85:395-405.
19. Yanagisawa S, Baker JR, Vuppusetty C, et al. Decreased phosphatase PTEN amplifies PI3K signaling andenhances proinflammatory cytokine release in COPD. Am J Physiol Lung Cell Mol Physiol 2017;313:L230-L239.
20. Gao W, Li L, Wang Y, et al. Bronchial epithelial cells: the key effector cells in the pathogenesis of chronic
obstructive pulmonary disease? Respirology 2015;20:722-729.
21. Global Strategy for the Diagnosis, Management and Prevention of COPD. Global Initiative for Chronic
Obstructive Lung Disease (GOLD) 2015. Available from: http://www.goldcopd.org/ .
22. Sriskantharajah S, Hamblin N, Worsley S, et al. Targeting phosphoinositide 3-kinase delta for the treatmentof respiratory diseases. Ann N Y Acad Sci 2013;1280:35-39.
23. Marwick JA, Chung KF, Adcock IM. Phosphatidylinositol 3-kinase isoforms as targets in respiratory disease.
Ther Adv Respir Dis 2010;4:19-34.
24. Kok K, Geering B, Vanhaesebroeck B. Regulation of phosphoinositide 3-kinase expression in health anddisease. Trends Biochem Sci 2009;34:115-127.
25. Ito K, Caramori G, Adcock IM. Therapeutic potential of phosphatidylinositol 3-kinase inhibitors in
inflammatory respiratory disease. J Pharmacol Exp Ther 2007;321:1-8.
26. Kim SJ, Jung HJ, Lim CJ. Reactive oxygen species-dependent down-regulation of tumor suppressor genes
PTEN, USP28, DRAM, TIGAR, and CYLD under oxidative stress. Biochem Genet 2013;51:901-915.
27. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2ARE pathway. Annu Rev Pharmacol Toxicol 2007;47:89-116.
28. Yamada K, Asai K, Nagayasu F, et al. Impaired nuclear factor erythroid 2-related factor 2 expression increases
apoptosis of airway epithelial cells in patients with chronic obstructive pulmonary disease due to cigarettesmoking. BMC Pulm Med 2016;16:27.
29. Church DF, Pryor WA. Free-radical chemistry of cigarette smoke and its toxicological implications. Environ
Health Perspect 1985;64:111-126.
30. Hoenderdos K, Condliffe A. The neutrophil in chronic obstructive pulmonary disease. Am J Respir Cell Mol
Biol 2013;48:531-539.
31. Hacievliyagil SS, Gunen H, Mutlu LC, et al. Association between cytokines in induced sputum and severity ofchronic obstructive pulmonary disease. Respir Med 2006;100:846-854.
32. Yamamoto C, Yoneda T, Yoshikawa M, et al. Airway inflammation in COPD assessed by sputum levels ofinterleukin-8. Chest 1997;112:505-510.
33. Di Stefano A, Capelli A, Lusuardi M, et al. Severity of airflow limitation is associated with severity of airway
inflammation in smokers. Am J Respir Crit Care Med 1998;158:1277-1285.
34. Stänescu D, Sanna A, Veriter C, et al. Airways obstruction, chronic expectoration, and rapid decline of FEV1
in smokers are associated with increased levels of sputum neutrophils. Thorax 1996;51:267-271.
35. Soler N, Ewig S, Torres A, et al. Airway inflammation and bronchial microbial patterns in patients with stablechronic obstructive pulmonary disease. Eur Respir J 1999;14:1015-1022.
36. Billadeau DD. PTEN gives neutrophils direction Nat Immunol. United States, 2008:716-718.
37. Zou W, Chu X, Cai C, et al. AKT-mediated regulation of polarization in differentiated human neutrophil-like
HL-60 cells. Inflamm Res 2012;61:853-862.
38. Yamada KM, Araki M. Tumor suppressor PTEN: modulator of cell signaling, growth, migration and apoptosis.
J Cell Sci 2001;114:2375-2382.

- 17 -

Osaka City Med. J. Vol. 65, 19-29, 2019

Focal Atrophy of the Dorsal Brainstem Correlates withHallucinations in Patients with Dementia with Lewy Bodies
TADASHI OHTOMO1), YASUNORI MATSUDA1), KENTARO UCHIDA1), JUMPEI MARUTA1), and KOKI INOUE1,2)
Departments of Neuropsychiatry1) and Center for Brain Science2),
Osaka City University Graduate School of Medicine

Abstract
Background
Visual hallucinations, the characteristic symptom of dementia with Lewy bodies (DLB), and theirunderlying mechanisms have not been researched extensively and their pathophysiology remains
unknown. Because the quality of hallucinations in patients with DLB and peduncular hallucinosis(PH) is similar, we investigated the relationship between focal atrophy of the dorsal brainstem andhallucinations in patients with DLB.
Methods
Brain magnetic resonance imaging was conducted in 24 DLB patients and data were analyzed
with the voxel-based specific regional analysis system for Alzheimer s disease (VSRAD) advance 2
software, which assesses the degree of focal atrophy of dorsal brainstem gray matter (GM) and whitematter (WM). Patients were divided into two groups in three different ways: GM atrophy, WM
atrophy, and relative atrophic degree of either GM or WM. The degree of hallucinations wasestimated using the Neuropsychiatric Inventory (NPI). Then, the VSRAD results and NPI scores
were analyzed.
Results
The mean NPI total score and hallucination score were significantly lower in the group with WM
atrophy (p＜0.01), whereas there were no significant differences between the groups divided on thebasis of the degree of GM atrophy. There was also a significant inverse correlation between the
degree of WM atrophy and the NPI hallucinations score (p＜0.01).
Conclusions
Our findings suggest that focal atrophy of the dorsal brainstem GM with relatively spared WM inpatients with DLB might cause visual hallucinations similar to PH. Further longitudinal studies
could elucidate the pathophysiology of hallucinations in patients with DLB.
Key Words: ‌Dementia with Lewy bodies; Visual hallucinations; Peduncular
hallucinosis; Brainstem atrophy; MRI

Received August 30, 2018; accepted November 27, 2018.
Correspondence to: Tadashi Ohtomo, MD, MFA.
Department of Neuropsychiatry, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-3821; Fax: +81-6-6636-0439
E-mail: dayouli@gmail.com- 19 -

Ohtomo et al

Introduction
Dementia with Lewy bodies (DLB) is the second most common cause of neurodegenerative
dementia after Alzheimer disease (AD)1). Complex or formed visual hallucinations (VH) are a corefeature of DLB2,3); these are also reported in patients with Parkinson s disease (PD)4). VH in DLB are
typically described as non-threatening images of people, animals, or objects that persist for seconds tominutes. The images may be vivid, colorful, and detailed; for example, the people and animals may
be smaller than normal size Lilliputian , and dressed in colorful costumes. Patients are usuallyaware of the images hallucinatory nature. However, night-time hallucinations can be accompaniedby confusion where patients tend to interact with their VH and wandering behavior and agitation arecommon 5-7) . The phenomenology of VH in DLB has been described in detail; however, its
pathomechanism has not been researched extensively.
Several morphological changes in the specific brain regions associated with VH in DLB have beenreported, including changes in the right inferior frontal gyrus8), pedunculopontine nucleus9), superiorparietal gyrus and precuneus 10), and right anterior insula 11). Many studies report that themorphological changes are gray matter (GM) atrophy, yet some argue that they are DLB with otherneurodegenerative diseases such as PD9) and AD11). At present, consensus on this issue has yet to bereached. This dispute might be due to difference in duration of the disease and different methods ofevaluating focal brain atrophy and VH.
Peduncular hallucinosis (PH) is a syndrome of hallucinations and brainstem symptoms that was
first reported in 1922 by the French neurologist Lhermitte. His case report described a 72-year-old
female who experienced strange VH that occurred late in the day, particularly at dusk. Although thepatient knew the images were not real, she would sometimes try to touch them. Her symptoms
suggested a focal infarct involving the midbrain and pons (left ptosis, total external ophthalmoplegia,
lateral pulsion when walking, right dysmetria, intention tremor, and a Babinski sign). The
hallucinations in the reported case were associated with insomnia and daytime somnolence12). In PH,
the images are vivid with colorful people, animals, or scenes. PH and VH in DLB have distinctive
common features. Hayashi reported that a patient recovering from immune-mediated brainstem
encephalitis was able to draw colorful, vivid pictures of his hallucinations13).
The quality of the VH in DLB closely resembles that of PH: complex, vivid, detailed, and usuallyoccur at dusk, as described above. The duration of hallucinations, however, is one of the differences.
Whereas PH usually lasts several days to weeks14), VH in DLB last longer. Janzen indicatedsimilarities in the phenomenology of VH in PD and PH9). We hypothesized that VH in DLB might
involve a dorsal brainstem lesion similar to PH, particularly focal atrophy, and therefore investigatedtheir relationship using the Neuropsychiatric Inventory (NPI) and voxel-based specific regional
analysis system for Alzheimer s disease (VSRAD) advance 2 software, which evaluates dorsal
brainstem atrophy.

Methods
Patients
Twenty-four patients (12 men, 12 women) who were diagnosed with probable DLB through clinicalexaminations, including brain magnetic resonance imaging (MRI) and NPI, were recruited from theoutpatient clinic of the Department of Neuropsychiatry in the Osaka City University Hospital
between July 2013 and January 2018. Diagnoses were assisted by 123iodine-metaiodobenzylguanidine- 20 -

Brainstem Atrophy and Hallucinations in DLB(MIBG) myocardial scintigraphy (4 cases), dopamine transporter (DAT) imaging (11 cases) or both (1case). The mean age of the patients was 81.0±6.1 years. The clinical diagnosis of DLB was based on
the third consensus report of the DLB consortium2). The patients who were suspected of AD s or
cerebrovascular dementia s complications were excluded by clinical interviews and examinations
including brain imaging mentioned above. Other causes of dementia and cognitive impairment,
including thyroid dysfunction, venereal diseases (including neural syphilis), and vitamin B12 or folate
deficiency, were excluded by laboratory investigations. This study was reviewed and approved by the
Human Subject Review Committee at Osaka City University, and informed consent was obtained
from each patient and a representative from his or her family after a detailed explanation of thisstudy was given.
Functional and neuropsychological assessment
Each patient underwent a clinical assessment before the MRI procedure. To assess the presenceof cognitive impairment, the Mini-Mental State Examination (MMSE)15) and Revised Hasegawa
Dementia Scale (HDS-R: from 0 to 30, with 0 being the most severe)16) were used. The HDS-R has
been used exclusively in East Asian countries as a screening test for dementia. The severity of thehallucinations was rated using the Japanese version of the NPI which assesses 10 sub-domains of
behavioral functioning, namely, delusions, hallucinations, agitation/aggression, dysphoria, anxiety,euphoria, apathy, disinhibition, irritability/lability, and aberrant motor activity. Each sub-domain
examines both frequency and severity and the score is obtained by multiplying these scores. The
frequency rating is from 0 to 4, and the severity rating is from 0 to 3. The total NPI score is the sumof the domain scores (from 0 to 120)17,18).
MRI procedure
All subjects underwent an MRI study using a 1.5T Vision Plus imager (Avanto, SIEMENS,
German). Axial, coronal, and sagittal T1-weighted sequence images and axial T2-weighted sequence
images were obtained for diagnosis. For VSRAD analysis, 3-D T1-weighted sequence imaging wasused [field of view (FOV), 230 mm; time of repeat (TR), 1900 ms; time to echo (TE), 3.14 ms; flip
angle, 25°; and phase resolution, 256, 1.00-mm slice thickness].
Imaging analysis
All MRI data were analyzed with VSRAD advance 2 ® (Eisai, Japan) software based on voxel-based
morphometry (VBM), an automated method for analysis of brain MRI images that statisticallyevaluates atrophy at the voxel level. This software program, which runs on Windows for VBM
analysis with Statistical Parametrical Mapping (SPM) and the Diffeomorphic Anatomical
Registration Through Exponentiated Lie algebra (DARTEL), was developed to differentiate DLB and
AD. 3D-T1-weighted whole brain images were spatially normalized with a 12-parameter affine
transformation to the SPM template so as to correct for differences in brain size. These linearlytransformed images were nonlinearly transformed and then modulated to the customized templatefor DARTEL, followed by smoothing using an 8-mm full width at half maximum (FWHM) kernel.
The GM and white matter (WM) image of a patient was compared with the mean and standard
deviation (SD) of GM images of healthy volunteers using voxel-by-voxel analysis. Finally, the z score
was acquired {z score＝[(control mean)-(individual value)]/control SD}. These z score maps weredisplayed by overlay on tomographic sections and surface renderings of the standardized brain. The
target volume of interest (VOI) included the entire region of the entorhinal cortex, hippocampus,
amygdala, and dorsal brainstem (Fig. 1). The validity of the VSRAD software has already been

- 21 -

Ohtomo et alreported elsewhere19). In the present study, we used the averaged positive z score in the GM and WMof the dorsal brainstem. The higher the z score, the more severe the atrophy in the target VOI.
Using VSRAD advance 2, the dorsal brainstem GM and WM z scores are automatically calculated.
The patients were divided into two groups in three different ways: GM atrophy, WM atrophy, andrelative atrophic degree of either GM or WM (subtracted value of GM from WM). We adopted themean z score of GM and WM, which were 0.491 and 0.611 respectively, as the criteria for the first andsecond grouping, and the subtraction value of zero (z score of GM and WM are equal) as the criterion
for the third.
Statistical analysis
Statistical analysis was performed with SPSS for Windows 23.0 (SPSS Japan, Tokyo, Japan). We

Figure 1. GM atrophy map (a) and WM atrophy map (b) of an octogenarian man with DLB. Colored areas indicating
higher z score are overlaid on tomographic sections of the patient s MRI. The target VOI in the dorsal brainstem
is demarcated with the yellow line in axial planes of standardized MRI template (c). GM, gray matter; WM, whitematter; DLB, dementia with Lewy bodies; MRI, magnetic resonance imaging; and VOI, volume of interest.

- 22 -

Brainstem Atrophy and Hallucinations in DLB

compared the clinical characteristics and VSRAD parameters between the two groups with the MannWhitney s U-test or Student s t-test according to the normality in distribution. Categorical variables
such as sex were analyzed using the chi-square test. The normality in distribution was examinedusing the Shapiro-Wilk test. The following statistical examinations were also used: Mann-Whitney s
U-test for duration of disease and the NPI scores, chi-square test for sex distribution, and Student s
t-test for all other analyses. In addition, we assessed the correlation between z score and NPI score

Table 1. ‌Demographic and clinical characteristics of the GMA and GMNA, WMA and WMNA groups, or
GM≧WM and WM≧GM atrophy groups
1a. Categorized on the basis of the degree of GM atrophy
Total(n＝24)

GMA group(n＝10)

GMNA group(n＝14)

p value

12/12

4/6

8/6

0.527/0.593#

Age (years, mean±SD)81.01±6.09

80.15±6.06

81.62±6.50

0.580

Onset age (years)

77.76±5.63

77.83±6.39

77.71±5.50

0.963

Duration of DLB (years)

3.25±3.07

2.33±2.18

3.91±3.61

0.230

Male/Female

Education (years)

10.90±2.78

9.75±2.66

11.82±2.79

0.122

HDS-R total score17.88±6.1418.80±6.70

17.21±6.12

0.553

MMSE total score

19.67±4.89

19.90±4.93

19.50±5.210.852

WMA group(n＝9)

WMNA group(n＝15)

p value

1b. Categorized on the basis of the degree of WM atrophy
Total(n＝24)
Male/Female

12/12

5/4

7/8

0.739/0.796#

Age (years, mean±SD)81.01±6.09

83.92±5.4479.27±6.17

0.076

Onset age (years)

77.76±5.63

80.86±4.2575.90±5.84

0.037 ＊

Duration of DLB (years)

3.25±3.07

3.06±2.52

3.37±3.54

0.818

Education (years)

10.90±2.78

10.71±2.63

11.08±3.09

0.795

HDS-R total score17.88±6.14

18.89±6.77

17.27±6.11

0.551

MMSE total score

19.67±4.89

20.44±5.13

19.20±5.03

0.566

1c. Categorized on the basis of the value of subtraction (WM-GM)
Total(n＝24)

GM≧WM group(n＝11)

WM≧GM group(n＝13)

p value

12/12

4/7

8/5

0.366/0.405#

Age (years, mean±SD)81.01±6.09

78.34±6.63

83.27±5.06

0.051

Onset age (years)

77.76±5.63

75.85±6.33

79.37±4.89

0.138Duration of DLB (years)

3.25±3.07

2.49±2.16

3.90±3.74

0.262

Education (years)

10.90±2.78

10.13±2.75

11.55±2.91

0.298

HDS-R total score17.88±6.14

18.18±6.21

17.62±6.56

0.831

MMSE total score

19.67±4.89

19.82±4.62

19.54±5.47

0.895

Male/Female

p value of GMA/GMNA, WMA/WMNA or GM≧WM/WM≧GM group; ＊p＜0.05.

#

GMA group, patients with gray matter atrophy; GMNA group, patients without gray matter atrophy; WMA group,
patients with white matter atrophy; WMNA group, patients without white matter atrophy; GM≧WM group, patientswith relatively severe gray matter atrophy; WM≧GM group, patients with relatively severe white matter atrophy;
GM, gray matter; WM, white matter; DLB, dementia with Lewy bodies; HDS-R, the Revised Hasegawa Dementia
Scale; and MMSE, Mini-Mental State Examination.

- 23 -

Ohtomo et alusing the Spearman rank correlation coefficient. All statistical tests were two-tailed, withsignificance at p＜0.05.

Results
Table 1 shows the demographic features of the patients with DLB categorized on the basis of thedegree of dorsal brainstem atrophy of GM (Table 1a), of WM (Table 1b), or of GM subtracted from WM(Table 1c). There were no significant differences in the age, onset age, duration of dementia, HDS-R
total score, or MMSE total score between the groups, except for onset age of the WMA (WM atrophy)
and WMNA (without WM atrophy) groups.
The NPI subscales showed no significant differences (data not shown) except for hallucinations
score. When the patients were divided using degree of WM atrophy, the NPI total and hallucinations
scores of the WMNA group were significantly higher than those of the WMA group (Table 2b),whereas there was no significant difference among the groups divided on the basis of the degree ofGM atrophy (Table 2a). In the groups divided using the subtraction (WM-GM) value, thehallucinations score of the GM≧WM (GM atrophy was relatively severe and WM was relatively
spared) group was higher (Table 2c). In the hallucinations subscale of NPI, acoustic, optic, andTable 2. ‌Differences in NPI score between the GMA and GMNA, WMA and WMNA groups, or GM≧WM and
WM≧GM atrophy groups
2a. Categorized on the basis of the degree of GM atrophy
GMA group(n＝10)

GMNA group(n＝14)

p value

Delusion (mean±SD)

2.70±3.69

3.86±3.18

0.291

Hallucination

5.00±3.58

3.14±3.66

0.197

Total score16.60±14.56

17.29±11.46

0.639

WMA group(n＝9)

WMNA group(n＝15)

p value

Delusion (mean±SD)

2.11±2.42

4.00±3.81

0.341

Hallucination

1.11±1.59

5.60±3.65

0.004 ＊＊

Total score

8.78±6.14

21.93±13.29

0.007 ＊＊

GM≧WM group(n＝11)

WM≧GM group(n＝13)

p value

Delusion (mean±SD)

3.45±3.70

3.31±3.22

1.000

Hallucination

6.36±3.57

1.85±2.38

0.004 ＊＊

Total score19.73±15.29

14.69±9.77

0.582

2b. Categorized on the basis of the degree of WM atrophy

2c. Categorized on the basis of the value of subtraction (WM-GM)

NPI, Neuropsychiatric Inventory; GMA group, patients with gray matter atrophy; GMNA group, patients without
gray matter atrophy; WMA group, patients with white matter atrophy; WMNA group, patients without white matter
atrophy; GM≧WM group, patients with relatively severe gray matter atrophy; WM≧GM group, patients withrelatively severe white matter atrophy; GM, gray matter; and WM, white matter. ＊＊p＜0.01.

- 24 -

Brainstem Atrophy and Hallucinations in DLB
Table 3. ‌Correlation of dorsal brainstem atrophy and NPI score
GM z score

Delusion(WM-GM) value

coefficient (ρ)

p valuecoefficient (ρ)

p valuecoefficient (ρ)

p value−0.1450.498−0.1240.562−0.039

0.856

0.350

0.094

−0.5210.009−0.6030.002 ＊＊

−0.1020.636−0.3940.057−0.312

0.138

Hallucination
Total score

WM z score＊＊

NPI, Neuropsychiatric Inventory; GM, gray matter; and WM, white matter. ＊＊ p＜0.01.

Figure 2. Correlation of the NPI hallucinations score and subtracted value of z score (WM z score-GM z score).
Smaller value indicates GM atrophy is relatively severe, and vice versa. NPI, Neuropsychiatric Inventory; WM, whitematter; and GM, gray matter.

olfactory hallucinations were rated together. Thus, we retrospectively checked the medical records
and confirmed that the main hallucinatory symptom of these patients was VH.
Correlation between the degree of atrophy in the dorsal brainstem and NPI score was examined.
Significant inverse correlations were observed in the WM atrophy and NPI hallucination scores (ρ＝−0.521, p＝0.009) and also in the WM-GM value and NPI hallucination score (ρ＝−0.603, p＝0.002)(Table 3 and Fig. 2).

Discussion
We examined VH and dorsal brainstem atrophy in patients with DLB using VSRAD advance 2
software which automatically assesses the degree of atrophy. The results of the present study suggestthat the correlation between brainstem GM atrophy and hallucinations is not obvious while WM
atrophy inversely correlates. Because the GM of the dorsal brainstem, especially that of the
midbrain, showed significant atrophy in the patients with DLB20), we speculate that impaired nuclei
with comparatively spared axons might cause disturbances in perception. In other words,
degenerating nerve cell bodies with a spared neural network provokes hallucinations. This idea

- 25 -

Ohtomo et al

seems to be consistent with previous studies arguing that the lesions related to PH areheterogeneously distributed, whereas the lesions show specific network anti-correlation with theextrastriae visual cortex21).
Our findings suggest that focal atrophy of brainstem GM with relatively spared corresponding
WM in patients with DLB might cause symptoms similar to PH. In the literature, the culprit lesionof PH includes infarction22-26), hemorrhage27), hematoma24), inflammation13), and tumor28-30). There arealso a few reports of PH as a side effect of cardiac or cerebral angiography26,31). We speculate that
dorsal brainstem GM atrophy with relatively retained WM might have a similar pathophysiology tothat of brainstem infarction, hemorrhage, hematoma, inflammation, and tumor.
In terms of the pathomechanism of PH, Lhermitte proposed the hypothesis that a hallucinationreflects a consequence of derangement in the sleep-wake cycle12,14). His contemporary neurologist, vanBogaert, on the other hand, proposed that hallucinations were a state of ego dissolution with loss ofability to distinguish external reality from imagination14,23,32). In recent years, there are few literatures
present pathophysiological models to explain PH. These models suppose that a loss of brainstem
control of the cortex is the primary factor of PH33). Middleton and Strick s model emphasizes functionof substantia nigra pars reticularis (SNpr) which has inhibitory effect to infero-temporal cortex
through thalamus. It has been previously shown that artificial stimulation at infero-temporal cortex
induces VH in humans 34). A lesion at SNpr removes suppression of the thalamus and as aconsequence leads to abnormal excitation of infero-temporal cortex accompanied by VH35). Manford
and Andermann s model focuses on dorsolateral geniculate nucleus (dLGN) which is under brainstem
control and act as a center for brainstem modulation of inputs from the retina to the visual cortex. It
is shown that dLGN receives many inputs from various neurons including pedunculopontine
tegmental nucleus (PPTN) which is the main component of the reticular activating system36). Given
that a loss of brainstem control of the cortex is the cause of PH, one of possible interpretations of ourresults might be as follows: impaired nuclei (GM) cause unfiltered or disordered signals and sparedaxons (WM) transmit to neural network. Consequently, the signal leads confusion and over-activity
in the cortex, in other words, a loss of control of the cortex. And if axons are atrophic and theirfunctions are lost, the disturbance to the neural network is relived.
However, the issue of the etiology of PH is still controversial today. Further detailed
investigations are needed to examine brainstem lesions of patients with PH. Morphological studies ofbrain used to focus on GM and tend to neglect WM. Our study indicates WM have profound influenceon the brain functioning. The authors hope that future study regarding not only GM but also WMmight deepen our understanding of underlying mechanism of hallucinations.
VSRAD advance 2 may be useful for objective assessment of atrophy at the dorsal brain stem inpatients with dementia and hallucinations. The software was originally designed to evaluate medial
temporal lobe atrophy in order to diagnose AD. Later, the VOI was extended so that the dorsal
brainstem , namely, the midbrain and pons, was included, because it was revealed that the medial
temporal lobe was relatively retained and the dorsal midbrain and pons were more atrophic inpatients with DLB20,37,38). Although the value of [(z score of dorsal brainstem atrophy)/(z score of
medial temporal atrophy)] is the index for AD-DLB differentiation, we adopted the raw z score of
dorsal brainstem atrophy as the index because we were not focused on the relationship with medial
temporal atrophy but on the extent of focal atrophy itself.
There are some limitations in the present study. First, the number of participants was relatively- 26 -

Brainstem Atrophy and Hallucinations in DLB

small (24 patients) and we should consider this as a pilot study. For further investigation andlongitudinal study, more patients should be recruited. Second, diagnoses of DLB was notpathologically confirmed but relied on clinical diagnoses alone; therefore, the possibility ofoverestimation cannot be excluded. Third, the influence of medication was not taken into
consideration. We used the data and information at the first visit of each participant, and mostpatients did not take anti-dementia medication. The influence of other medication, however, isundeniable. Fourth, although visual disturbances are reported as one of the hypothesized causes of
VH in PD and DLB39,40), our patients did not undergo ophthalmological testing. On the other hand,
despite the occurrence of visual abnormalities, ocular pathology does not seem to distinguish thepatients who hallucinate from those who did not41). However, ophthalmological examination results
should be taken into consideration in future studies. Fifth, although VSRAD enable us to assess
atrophy objectively, the target VOI is fixed and it is not possible to tell which region is atrophic within
the VOI. In further investigations, morphological change should be examined with other method
which is able to intentionally set VOI. The regions to examine preferably include the occipital lobe
that is thought to be related VH, brainstem or neighboring nuclei such as SNpr, PPTN and dLGN,
and projecting fibers between these structures.
Finally, our result leads to the further presumption that hallucinatory symptoms may alleviate as
WM atrophy progresses with the natural process of neural degeneration. These results are consistent
with Braak s hypothesis indicating that physical contact between susceptible regions may play a keyrole and that α-synuclein inclusions developed from the olfactory bulbs to the neocortex via themedulla oblongata and midbrain42). In terms of our participants background, the WM atrophy grouptended to be older, although this difference was not significant (Table 1b and 1c), and onset age of theWM atrophy group was statistically higher (Table 1b). Therefore, VH in DLB seems to be relieved by
the passage of time. It is also consistent with the common clinical course of VH in DLB.
Hallucinations usually do not exacerbate, but gradually alleviate over time. Although we tend toassume that the remission of VH is the effect of medical treatment, including drug therapy, it might
partially be the natural course of DLB.
Acknowledgments
All authors have no COI to declare regarding the present study.
The authors would like to thank R. Tagawa for valuable discussions. We also thank H. Ohtomo
for his help in reading French articles. Finally, we are grateful to the referees for useful comments.
References
1. Galasko D, Hansen LA, Katzman R, et al. Clinical-neuropathological correlations in Alzheimer s disease andrelated dementias. Arch Neurol 1994;51:888-895.
2. McKeith IG, Dickson DW, Lowe J, et al. Diagnosis and management of dementia with Lewy bodies: third
report of the DLB Consortium. Neurology 2005;65:1863-1872.
3. McKeith IG, Boeve BF, Dickson DW, et al. Diagnosis and management of dementia with Lewy bodies: fourthconsensus report of the DLB Consortium. Neurology 2017;89:88-100.
4. Fénelon G, Mahieux F, Huon R, et al. Hallucinations in Parkinson s disease: prevalence, phenomenology andrisk factors. Brain 2000;123:733-745.
5. Iseki E, Marui W, Nihashi N, et al. Psychiatric symptoms typical of patients with dementia with Lewy bodies similarity to those of levodopa-induced psychosis. Acta Neuropsychiatr 2002;14:237-241.
6. Nagahama Y, Okina T, Suzuki N, et al. Classification of psychotic symptoms in dementia with Lewy bodies.
Am J Geriatr Psychiatry 2007;15:961-967.

- 27 -

Ohtomo et al
7. Onofrj M, Taylor JP, Monaco D, et al. Visual hallucinations in PD and Lewy body dementias: old and new
hypotheses. Behav Neurol 2013;27:479-493.
8. Sanchez-Castaneda C, Rene R, Ramirez-Ruiz B, et al. Frontal and associative visual areas related to visual
hallucinations in dementia with Lewy bodies and Parkinson s disease with dementia. Mov Disord 2010;
25:615-622.
9. Janzen J, van t Ent D, Lemstra AW, et al. The pedunculopontine nucleus is related to visual hallucinations in
Parkinson s disease: preliminary results of a voxel-based morphometry study. J Neurol 2012;259:147-154.
10. Delli Pizzi S, Franciotti R, Tartaro A, et al. Structural alteration of the dorsal visual network in DLB patientswith visual hallucinations: a cortical thickness MRI study. PLoS One 2014;9:e86624.
11. Blanc F, Noblet V, Philippi N, et al. Right anterior insula: core region of hallucinations in cognitive
neurodegenerative diseases. PLoS One 2014;9:e114774.
12. Lhermitte J. Syndrome de la calotte du pédoncule cérébral. Les troubles psycho-sensoriels dans les lésions dumésocéphale. Rev Neurol 1922;38:1359-1365.
13. Hayashi Y, Yoshikura N, Kimura A, et al. Peduncular hallucinations in brainstem encephalitis drawn by apatient. Neurology 2012;79;1625.
14. Risser AH, Powell FC. Lhermitte s peduncular hallucinosis. In: 45th Annual Meeting of the American
Academy of Neurology, New York; 1993.
15. Folstein MF, Folstein SE, McHugh PR. Mini-mental state . A practical method for grading the cognitive stateof patients for the clinician. J Psychiatr Res 1975;12:189-198.
16. Kim KW, Lee DY, Jhoo JH, et al. Diagnostic accuracy of mini-mental status examination and revised
Hasegawa dementia scale for Alzheimer s disease. Dement Geriatr Cogn Disord 2005;19:324-330.
17. Cummings JL, Mega M, Gray K, et al. The Neuropsychiatric Inventory: comprehensive assessment of
psychopathology in dementia. Neurology 1994;44:2308-2314.
18. Hirono N, Mori E, Ikejiri Y, et al. Japanese version of the Neuropsychiatric Inventory-a scoring system forneuropsychiatric disturbance in dementia patients. No To Shinkei 1997;49:266-271. (In Japanese)
19. Matsuda H, Mizumura S, Nemoto K, et al. Automatic voxel-based morphometry of structural MRI by SPM8
plus diffeomorphic anatomic registration through exponentiated Lie algebra improves the diagnosis ofprobable Alzheimer disease. AJNR Am J Neuroradiol 2012;33:1109-1114.
20. Whitwell JL, Weigand SD, Shiung MM, et al. Focal atrophy in dementia with Lewy bodies on MRI: a distinct
pattern from Alzheimer s disease. Brain 2007;130:708-719.
21. Boes AD, Prasad S, Liu H, et al. Network localization of neurological symptoms from focal brain lesions. Brain
2015;138:3061-3075.
22. McKee AC, Levine DN, Kowall NW, et al. Peduncular hallucinosis associated with isolated infarction of thesubstantia nigra pars reticulata. Ann Neurol 1990;27:500-504.
23. Kölmel HW. Peduncular hallucinations. J Neurol 1991;238:457-459.
24. Benke T. Peduncular hallucinosis: a syndrome of impaired reality monitoring. J Neurol 2006;253:1561-1571.
25. Penney L, Galarneau D. Peduncular hallucinosis: a case report. Ochsner J 2014;14:450-452.
26. Notas K, Tegos T, Orologas A. A case of peduncular hallucinosis due to a pontine infarction: a rare complicationof coronary angiography. Hippokratia 2015;19:268-269.
27. Harada Y, Ishimitsu H, Miyata I, et al. Peduncular hallucinosis associated with ruptured basilar-superior
cerebellar artery aneurysm: case report. Neurol Med Chir (Tokyo) 1991;31:526-528.
28. Dunn DW, Weisberg LA, Nadell J. Peduncular hallucinations caused by brainstem compression. Neurology 1983;
33:1360-1361.
29. Nadvi SS, Ramdial PK. Transient peduncular hallucinations secondary to brain stem compression by a
cerebellar pilocytic astrocytoma. Br J Neurosurg 1998;12:579-581.
30. Biswas SN, Biswas S, Chakraborty PP. Peduncular hallucinosis as first presentation of juvenile pilocytic
astrocytoma. Indian J Pediatr 2016;83:736-737.
31. Rozanski J. Peduncular hallucinosis following vertebral angiography. Neurology 1952;2:341-349.
32. van Bogaert L. L hallucinose pédonculaire. Rev Neurol 1927;47:608-617.
33. Müri RM. Thalamic and brainstem regulatory systems - why disturbances external to the visual system cancause hallucinations. In: Collerton D, Mosimann UP, Perry E, editors. The neuroscience of visual
hallucinations. West Sussex: John Wiley & Sons; 2015. pp. 255-279.
34. Penfield W, Perot T. The brain s record of auditory and visual experience. a final summary and discussion.
Brain 1963;86:595-696.
35. Middleton FA, Strick PL. The temporal lobe is a target of output from the basal ganglia. Proc Natl Acad Sci U
S A. 1996;93:8683-8687.
36. Manford M, Andermann F. Complex visual hallucinations. Clinical and neurobiological insights. Brain 1998;- 28 -

Brainstem Atrophy and Hallucinations in DLB
121:1819-1840.
37. Kantarci K, Ferman TJ, Boeve BF, et al. Focal atrophy on MRI and neuropathologic classification of dementia
with Lewy bodies. Neurology 2012;79:553-560.
38. Nakatsuka T, Imabayashi E, Matsuda H, et al. Discrimination of dementia with Lewy bodies from Alzheimer sdisease using voxel-based morphometry of white matter by statistical parametric mapping 8 plus
diffeomorphic anatomic registration through exponentiated Lie algebra. Neuroradiology 2013;55:559-566.
39. Maurage CA, Ruchoux MM, de Vos R, et al. Retinal involvement in dementia with Lewy bodies: a clue tohallucinations. Ann Neurol 2003;54:542-547.
40. Matsui H, Udaka F, Tamura A, et al. The relation between visual hallucinations and visual evoked potential
in Parkinson s disease. Clin Neuropharmacol 2005;28:79-82.
41. Gallagher DA, Parkkinen L, O Sullivan SS, et al. Testing an aetiological model of visual hallucinations in
Parkinson s disease. Brain 2011;134:3299-3309.
42. Braak H, Del Tredici K. Nervous system pathology in sporadic Parkinson disease. Neurology 2008;70:19161925.

- 29 -

Osaka City Med. J. Vol. 65, 31-40, 2019

Age-specific Differences in Insulin dose of Japanese Patients withType 1 Diabetes of Various Ages
YUKO HOTTA1), TOMOYUKI KAWAMURA1), NAOKO NISHIKAWA1), KAYAKO HASHIMURA1,2),
YONEO KASHIHARA1,3), TOMOMI HASHIMOTO1,4), MASAKAZU HIROSE1,5), TAKASHI HIGASHIDE1,4),
HARUO SHINTAKU1), and TAKASHI HAMAZAKI1)
Department of Pediatrics1), Osaka City University Graduate School of Medicine;，
Hashimura Clinic2); Watamachi Kids Clinic3); Hug Hug Kids Clinic4); and D Medical Clinic Osaka5)

Abstract
BackgroundFew studies have investigated changes in insulin dose of Japanese patients with type 1 diabetes
with regard to patients age. Therefore, this study aimed to investigate it of the patients who wereusing insulin pump to help adjust each patient s insulin setting.
Methods
This study included 35 Japanese patients with type 1 diabetes aged ＜45 years who used Minimed
620G®, a type of insulin pump. Data on pump settings were retrospectively evaluated using theCarelink® system, which analyze data of insulin pump and continuous glucose monitor. We evaluatedtotal daily insulin dose per body weight, total daily basal insulin dose per total daily dose, thepatterns of basal insulin rate at each hour, carbohydrate ratio, and correction factor. We also
compared carbohydrate ratio or correction factor of our data with the number calculated by the 300
rule (carbohydrate ratio＝300/total daily insulin dose) or 1800 rule (correction factor＝1800/total daily
insulin dose).
Results
Age-specific tendency of insulin settings were turned out. One of the most remarkable results wasthat some preschool children needed a lower carbohydrate ratio than that calculated by the 300 ruleand a higher correction factor than that calculated by the 1800 rule.
Conclusions
Our data might help determine the initial insulin settings and adjust it for Japanese patients with
type 1 diabetes. Whereas, the rules which had been already reported cannot be applied to somepatients especially preschool children, and it is important that the insulin settings were individually
adjusted watching the blood glucose level.
Key Words: ‌Type 1 diabetes; Insulin pump; Carbohydrate ratio; Correction factor
Received September 3, 2018; accepted November 27, 2018.
Correspondence to: Tomoyuki Kawamura, MD.
Department of Pediatrics, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka, 545-8585, Japan
Tel: +81-6-6645-3816; Fax: +81-6-6636-8737
E-mail: kawam@med.osaka-cu.ac.jp- 31 -

Hotta et al

Introduction
Insulin pump is an insulin infusion device mainly used by type 1 diabetes patients. In the insulin
pump therapy, insulin pumps deliver rapid- or short-acting insulin 24 hours a day through a catheter
placed under the skin. The insulin doses are categorized as basal and bolus insulin.
Basal insulin is continuously delivered over 24 hours and maintains blood glucose levels in thenormal range between meals and overnight. Different amounts of insulin can be programmed at
different times (basal rate) of the day and night.
When patients eat, they use the insulin pump to administer additional insulin doses, called bolus,
to cover the carbohydrate content in each meal or snack; this is known as carb bolus. Patients also
take a bolus to treat high blood glucose levels, which is known as correction bolus. If patients havehigh blood glucose levels before eating, correction or supplemental bolus of insulin is required toreduce the levels to their target range1,2）.
Carbohydrate counting calculates the number of bolus insulin units required with respect to foodintake. Carbohydrate ratio (CR) calculates how many grams of carbohydrate one unit of insulin
will cover for in a meal. Correction factor (CF) calculates how much decrease in blood sugar level(mg/dL) will one unit of insulin lead to at hyperglycemia. The effects of carbohydrate counting for
adjusting insulin doses have been reported 3,4). In Japan, carbohydrate counting has recently become
popular. Some rules, such as the 500 rule and 1800 rule, have been reported to calculate CR and CF
of patients. In the 500 rule, CR is calculated by dividing 500 by the total daily dose (TDD) of insulin.
Some researchers reported that the 300 or 400 rule was better than the 500 rule5,6). Furthermore, inthe 1800 rule, CF is calculated by dividing 1800 by TDD, and some researchers have recommended
the 1700 rule for CF7).
Insulin doses of type 1 diabetes patients change as their age. To understand the trend of
insulin dose of type 1 diabetes patients with respect to age is useful for adjusting each patient s
insulin settings. Although some studies have reported on the number of insulin units requiredby patients3,5,6,8), but few studies have investigated the insulin dose for type 1 diabetes Japanesechildren and adolescents. Kuroda et al and Nakamura et al respectively reported that basal insulin
requirement was around 30% of TDD in Japanese patients and it was lower rate than Western pastreports8,9), but the subjects were adult. There was a possibility that not only age-specific differences
but also racial differences in insulin settings. It was necessary to investigate Japanese children andadolescents.
This study aimed to investigate age-specific differences in basal rate, CR, and CF of Japanesepatients with type 1 diabetes and assess whether it was consistent with the above rules to help
determine and adjust insulin settings for each patient.

Methods
The subjects of this study were type 1 diabetes patients who were using Minimed 620G®, a insulin
pump with continuous glucose monitor (CGM). The CGM is a revolutionary technology developedin the last two decades that continuously measures glucose levels in subcutaneous tissues via aninserted sensor. Patients with diabetes and medical staffs need to be able to adjust insulin and/or drug regimens. The sensor augmented pump (SAP) is an insulin pump that displays CGM data.
Minimed 620G ® is the first SAP which could be used in Japan since 2015. Using SAP, patients
can adjust their insulin dose according to CGM data, and doctors can analyze CGM data using the- 32 -

Age-specific Differences of Insulin Pump Settings

Carelink® system, the system analyzing data of insulin pump and continuous glucose monitor, andmore accurately adjust insulin doses than before10). On the other hand, it is possible to use Minimed
620G® without CGM, and some patients in this study sometimes used the pump without CGM.
Type 1 diabetes was diagnosed according to the criteria for acute onset type 1 diabetes proposedby the Japanese Diabetes Society11). Minimed 620G® were introduced to the type 1 diabetes patientsat the Department of Pediatrics of Osaka City University Hospital who were judged that Minimed
620G® were indicated by the doctor and who themselves wanted them. There were 87 Japanesepatients with type 1 diabetes aged ＜45 years who had visited the hospital and had used Minimed
620G® for ＞2 months in March of 2017. We excluded the patients as follows from the 87 patients;(1) 2 patients with advanced diabetic chronic complications, (2) 3 pregnant women, (3) 1 patient withmyositis taking steroid therapy, (4) 1 patient with short stature taking growth hormone therapy, (5)8 patients whose blood C-peptide levels were ＞0.6 ng/mL, (6) 4 patients who were not using bolus
calculator, the system of insulin pump which calculates unit of bolus by inputting the blood glucose
level and the amount of carbohydrate they were going to eat if CR and CF were registered in advance.
The data of the remaining 68 patients during January and March 2017 were examined, but thedata of 33 patients were missing for some reason, such as self-discontinuation of visiting the hospital,
computer trouble, and so on.
Finally, 35 subjects were enrolled (male/female: 8/27, aged 4.4-41.8 years). Blood C-peptide levels
were undetectable in 24 patients and were 0.02-0.31 ng/mL in the other 11 patients. All patientsused rapid-acting insulin. Two patients in the preadolescent group used long-acting insulin via thepen delivery system (one used glargine U300 and the other used degludec) to administer basal insulin
so as to avoid wearing the insulin pump during sports activity.
The patients were classified into the following four groups by age: preschool ( ＜6 years, n＝8),

Table 1. Characteristics of the subjects
Preschool

Preadolescent

Adolescent

Adults

n＝8

n＝14

n ＝8

n ＝5

Sex (male/female)

4/4

3/11

1/7

0/5

Age (years)

6.2

10.8

14.8

34.9

(4.4-6.7)(7.2-12.6)(13.4-20.8)(33.4-41.8)

8.6

8.0

8.0

7.3

(7.1-9.2)(6.7-8.9)(6.0-9.1)(6.9-7.8)

2.9

5.0

10.5

26.1

(1.5-4.7)(2.0-8.5)(1.8-15.7)(16.8-29.3)

4 (50.0%)

4 (28.6%)

2 (25.0%)

1 (20.0%)(0.05, 0.07, 0.1, 0.25)(0.02, 0.13, 0.25, 0.28)(0.02, 0.31)(0.18)

0.8

0.2

0.7

(−1.1-1.6)(−1.0-0.9)(−0.1-2.3)

HbA1c (%)
T1DM duration (years)
Number of patients whose
CPR were detected
BMI-SDS
BMI (kg/m )

26.2

2

(21.5-29.1)
The data were shown as a median (range). The C-peptide (CPR) was occasional serum C-peptide (ng/mL). TIDM, type1 diabetes; CPR, C-peptide; BMI, body mass index; and BMI-SDS, BMI standard deviation score.

- 33 -

Hotta et alpreadolescent (7-12 years, n＝14), adolescent (13-20 years, n＝8), and adults (33-41 years, n＝5). No
patients aged 21-32 years used the Minimed 620G®.
Patients themselves adjusted their insulin settings daily according to continuous glucose monitor(CGM) and self-monitoring of blood glucose (SMBG) data. They visited the hospital once each month,where their CGM data and pump records were checked by doctors using the Carelink® system, andtheir insulin settings were adjusted. Basal insulin dose was adjusted so that the patient s blood
glucose level was not changed when food was not consumed. CR was adjusted so that blood glucose
levels 4 hours after consuming a meal returned to pre-meal blood glucose levels when hyperglycemia
was not observed before meals. CF was adjusted to reduce high blood glucose levels to target blood
glucose levels 4 hours after the bolus injection when no food was consumed. In this study, insulin
doses were retrospectively evaluated using the Carelink® system from January to March 2017. The
study was approved by Ethics Committee of Osaka City University (authorization number: 3743).
We evaluated the median total daily doses (TDD/kg), the median percentage of total daily basal
insulin dose to total daily dose (TBD/TDD), the basal insulin rate at each hour, CR, and CF. We also
evaluated those were different by age. We calculated CR×TDD to evaluate whether 500 rule (CR＝500/TDD) or 300 rule (CR＝300/TDD) that had been recommended were applicable. Similarly, we

calculated CF×TDD to evaluated whether 1800 rule (CF＝1800/TDD) or 1700 rule (CF＝1700/TDD).
The results were analyzed by single regression analysis, distributed analysis by iterative
measurement, and the Kruskal-Wallis test using IBM SPSS Statistics, version 24. The KruskalWallis test was used to compare among groups, and the distributed analysis by iterative measurement
was used when considering difference in time. P values ＜0.05 were considered significant.

Results
Thirty-five subjects were enrolled in this study. The proportion of female was higher in preschool,
adolescent, and adult. Median HbA1c of preschool group was higher than other groups. We
thought that this meant the difficulty of blood glucose control in patients of that age, and it was alsocharacteristic of this age. Some reports have reported that HbA1c in this population is higher thanother ages12,13). Therefore, we thought that this group of subjects in our study were not a special group(Table 1).
The median total daily doses (TDDs) per body weight were 0.79 (0.56-0.91), 1.00 (0.58-1.71), 0.93
(0.72-1.21), and 0.65 (0.60-1.16) unit/kg in the preschool, preadolescent, adolescent, and adult groups,
respectively, described as median (range). The median TDDs seemed to increase with age until
preadolescence or adolescence age, after which they appeared to decrease, although not significantly(Fig. 1).
The median percentage of total daily basal insulin dose (TBD) to TDD (TBD/TDD) was 32.8%(18.2%-55.3%) (Fig. 2). Two patients in the preadolescent group who used long-acting insulin were
excluded from this analysis. It was evaluated by single regression analysis and no age-specificdifferences were observed (p＝0.948). There was no significant relationship between TBD/TDD andsex (p＝0.48) or TDD (p＝0.682). Patients of preschool, preadolescent, and adolescent groups were
evaluated using the body mass index standard deviation score (BMI-SDS), whereas those of adultgroup were evaluated based on BMI, and TBD/TDD was not associated with BMI or BMI-SDS.
The patterns of basal insulin rate at each hour appeared to be different among the groups. We
evaluated the basal ratio of every hour (%) to total basal dose (Fig. 3). Children and adolescents- 34 -

Age-specific Differences of Insulin Pump Settings

Figure 1. Relationship between age and TDD/kg. The broken line shows an approximate line. TDD, total daily dose.

Figure 2. Relationship between age and TBD/TDD. The broken line shows an approximate line. TBD, total daily
basal insulin dose; and TDD, total daily dose.

seemed to require higher basal insulin rates during 03:00-08:00, but not significantly in this study. In
preschool and preadolescent children, the basal rate seemed to be higher in the morning and evening
and lower in the daytime. These patterns were not related to whether C-peptide levels of the patientswas positive or negative.
The median CRs were 16.7 (11.1-25.0), 14.3 (5.0-50.0), 7.0 (5.9-8.3), and 7.7 (3.6-10.0) g/unit in- 35 -

Hotta et al

Figure 3. Basal infusion rate of the time zone in different groups. The data was shown the percentage of basal
infusion dose every hour in total basal dose among groups.

Table 2. Carbohydrate ratio and correction factor

Preschool
Preadolescent
Adolescent
Adult

Breakfast

Lunch

DinnerTotal

CR

14.3(11.1-30.3)

16.7 (11.1-25.0)

14.3 (11.1-25.0)

16.7(11.1-25.0)

CF

135 (100-300)

135 (100-400)

130 (100-300)

135 (100-400)

CR

11.3 (5.0-30.3)

13.4 (5.0-33.3)

14.7 (5.0-50.0)

14.3 (5.0-50.0)

CF

60 (30-300)

55 (30-300)

55 (30-300)

55 (30-300)

CR

6.7 (5.9-8.3)

6.0 (5.0-7.7)

6.3 (5.0-8.0)

7.0 (5.9-8.3)

CF

40 (30-60)50 (30-60)

40 (250-60)50 (30-60)

CR

7.7 (4.9-9.1)

7.7 (2.9-10.0)

7.7 (3.1-10.0)

7.7 (3.6-10.0)

CF

42.5 (20-50)

42.5 (20-50)

42.5 (20-50)

42.5 (20-50)

The data were shown as a median (range). CR and CF were picked up from Carelink system, the system analyzing
data of insulin pump and continuous glucose monitor, after they were adjusted. CR, carbohydrate ratio; and CF,
correction factor.
®

the preschool, preadolescent, adolescent, and adult groups, respectively (Table 2). Fourteen patients(40.0%) required lower CR for breakfast than for lunch or dinner. In particular, 50.0% and 57.1% of
patients in the preschool and preadolescent groups, respectively, required lower CR for breakfast thanthat for lunch or dinner (This result was not included in the table or figure). CRs of six patients (75.0%)
in the preschool group were lower than those calculated by the 300 rule (Fig. 4). We calculated
the constant, by the formula of CR×TDD in our data to compare that to 300-500 rules which were
proposed in previous reports. As the results, for breakfast, 218.6 (142.9-534.8) in preschool, 365.6(282.5-790.9) in preadolescents, 368.2 (248.5-392.0) in adolescents, and 328.5 (300.0-382.1) in adults.
For lunch, 234.3 (146.7-532.5) in preschool, 427.4 (334.0-557.5) in preadolescents, 358.6 (290.2-404.6)
in adolescents, 328.5 (228.9-402.0) in adults. For dinner, 234.3 (146.7-441.3) in preschool, 413.3(226.0-680.0) in preadolescents, 331.8 (276.7-375.7) in adolescents, 314.7 (250.3-402.0) in adults. To- 36 -

Age-specific Differences of Insulin Pump Settings

Figure 4. Relationship between TDD and CR or CF. The solid line of the 500 rule and the broken line of the 300 rule
were shown to be compared with the patient s CR. The solid line of the 1800 rule and the broken line of the 1700 rule
were shown to be compared with the patient s CF. CR or CF of all patients except preschool children were plotted
as dots and CR or CF of preschool children were especially plotted as diamond marks. TDD, total daily dose; CR,
carbohydrate ratio; and CF, correction factor.

summarize, at this study, the constant of many patients, CR×TDD was 300-450 in except preschool
children and 200-250 in preschool children especially at morning.
The median CFs were 135 (100-400), 55 (30-300), 50 (30-60), and 42.5 (20-50) mg/dL/unit in thepreschool, preadolescent, adolescent, and adult groups, respectively (Table 2). In many patients, CFs
did not differ with respect to the time of day. Many patients needed higher CF than those calculatedby the 1800 or 1700 rule (Fig. 4). CFs of eight patients in the preschool and preadolescent groups
were higher than 3000 divided by TDD; however, the relationship between CF and age remained
unclear. We calculated the constant, by the formula of CF ×TDD in our data to compare that to 17001800 rules which were proposed in previous reports. As results, CFs were 2034 (1376-4260), 2148
(1356-5010), 2355 (1494-2940), and 1617 (1602-2675) in the preschool, preadolescent, adolescent, andadult groups, respectively.

Discussion
In this study, changes in insulin doses of Japanese patients with type 1 diabetes with respect toage were investigated.
We observed that median TDDs seemed to increase with age until preadolescence or adolescenceage, after which they appeared to decrease, although not significantly among all groups. Lau et alreported that TDD/kg was 0.65 (0.61-0.70) in preschool group, 0.74 (0.62-0.83) in preadolescent, and0.88 (0.74-1.00) in adolescent, as mean±SD14). Danne et al reported that 0.71±1.31 in preschoolgroup, 0.73±0.16 in preadolescent group, and 0.83±0.19 in adolescent group15). They both reported
that TDD increased with age until adolescence age, which is similar to our study results.
The median TBD/TDD was 32.8% (18.2%-55.3%). The median TBD/TDD was previously
recommended to be approximately 50% of TDD in Western countries16). King et al recently proposed- 37 -

Hotta et ala new formula for calculating TBD: 0.4 ×TDD＝TBD17). The reason why a lower TBD value wasrecommended than previously used may be because the use of CGM became popular and basal dose
could be more accurately adjusted. Kuroda et al reported that TBD was under 30% of the TDD in
Japanese patients with type 1 diabetes8), and Nakamura et al reported that TBD to be 27.3%±6.0%
of TDD6). Generally, TBD/TDD was lower in Japanese data than in Western data. Hashimoto et alreported that in type 1 diabetes patients aged ≧14 years, TBD/body weight may be associated with
fat energy ratio of the meal18). In our study, fat energy ratio of the meal was not assessed; however,compared with Western food, Japanese food generally contains large amounts of carbohydrate,possibly resulting in Japanese patients requiring less basal insulin dose18).
However, TBD/TDD in our study was a little higher than that reported by Kuroda et al8) andNakamura et al6) in Japanese patients. There are several reasons for this discrepancy. One is thedifference in the ages of the subjects included in the studies. The subjects in their studies were
adults, whereas most of the subjects in our study were children and adolescents. Another reason
could be that our data were obtained from outpatients, whereas their data were obtained from
inpatients, and basal insulin settings can be more accurately adjusted in admitted settings.
In Japan compared with in Western country, TDD/kg was similar and TBD/TDD was less, so theinsulin dose of bolus was inferred to be higher. It might be also related to the higher carbohydrates
in Japanese meals.
Basal insulin patterns at each hour appeared to be different among the groups. We evaluatedbasal ratio of every hour to total basal dose. Children and adolescents seemed to need higher basal
insulin rates during 03:00-08:00. In preschool and preadolescent children, the basal rates seemed
to be higher in the morning and evening and lower in the daytime. The patterns of the basal rateaccording to time observed in our study were similar to those observed in previous studies14,19,20).
The tendency of increasing insulin requirements in the early morning was most prominent inadolescents, possibly because of the secretion pattern of growth hormone or cortisol early in themorning, known as the dawn phenomenon 21). The higher basal rates in the evening were observed inpreschool children, and preadolescent children; these findings are consistent with those previously
reported14,19,20). Although the mechanism underlying this phenomenon was unclear, one reason could
be the increase in growth hormone secretion22,23).
Fourteen patients (40.0%) had smaller CRs for breakfast than for lunch or dinner. Especially,
50.0% and 57.1% of patients in the preschool and preadolescent groups, respectively, had smaller CRs
for breakfast than for lunch or dinner, possibly because of cortisol secretion pattern in the morning,which increases insulin resistance in the morning24).
Kuroda et al reported that the CR was estimated by the 300 rule at breakfast or by the 400 rule
at lunch or dinner5), whereas Nakamura et al reported that the CR was estimated by the 300 rule atbreakfast, 500 rule at lunch, and 400 rule at dinner6). In our study, the calculated constant for CR
was 300-450 in all groups, except the preschool group, and 200-250 in the preschool group, especiallyin the morning. However, the reason why young children required smaller CRs for meal than otherage children was unclear. Their TDDs were related to their body weight. However, the nutritional
requirements did not linearly relate to the body weight of young children25). The discrepancy that
TDD was related to the body weight of young children, whereas energy requirement was not, may
explain the reason why their CRs were not calculated by the 300 rule, which is adaptable for older
patients.

- 38 -

Age-specific Differences of Insulin Pump Settings

Regarding median CFs, there was no difference with regard to the time of the day in many
patients. Furthermore, there was no correlation between CFs and age or TDD. The CFs of preschool
children varied more widely. The CFs of some preschool and preadolescent children were higher than3000 divided by TDD. The reason of this was unclear, but one possibility was that younger children
might have higher insulin sensitivity. The fact that TDD/kg of younger children was lower than older
patients might be also associated with their higher insulin sensitivity.
This study had some limitations. First, in this study, a meal omission test was not conducted for
adjusting basal insulin. This study was a retrospective study, and it was difficult for children to omit
meal. Meal omission tests were not conducted in some previous studies whose subjects were children
and adolescents14,15). Second, we could not determine whether the calculated CRs and CFs werecorrect. Therefore, further studies should assess the accuracy of our results.
In this study, we assessed whether the rules, which have been already reported for the calculationof CR and CF, could be applied to children and adolescents with type 1 diabetes or not. As a result,
those rules were not suitable to some patients especially preschool children. The rules used only TDDof the subjects. However, age and TDD among the subjects showed a complex relationship as shownin Figure 1. The complex relationship between age and TDD might be a reason why the rules could
not be applied to our subjects.
In conclusion, this study revealed the age-related differences in insulin dose and settings for pumptherapy. We believe that our data might be helpful in determining the initial insulin settings andadjusting it for Japanese patients with type 1 diabetes. On the other hand, the rules which have been
already reported cannot be applied to some patients especially preschool children, and it is importantthat the insulin settings were individually adjusted watching the changes of blood glucose level.
Acknowledgments
All authors have no COI to declare regarding the present study.
References
1. Subramanian S, Baidal D, Skyler JS, et al. The Management of Type 1 Diabetes. In Endotext. Edited by De
Groot LJ, Chrousos G, Dungan K, et al. MDText.com, Inc.; 2000.
2. Matejko B, Kukulka A, Kiec-Wilk B, et al. Basal insulin dose in adults with type 1 diabetes mellitus on insulin
pumps in real-life clinical practice: a single-center experience. Adv Med 2018;2018:1473160.
3. Göksen D, Atik Altinok Y, Ozen S, et al. Effects of carbohydrate counting method on metabolic control inchildren with type 1 diabetes mellitus. J Clin Res Pediatr Endocrinol 2014;6:74-78.
4. Yamada E, Okada S, Nakajima Y, et al. Effect of carbohydrate counting using bolus calculators on glycemic
control in type 1 diabetes patients during continuous subcutaneous insulin infusion. J Diabetes Investig 2017;
8:496-500.
5. Kuroda A, Yasuda T, Takahara M, et al. Carbohydrate-to-insulin ratio is estimated from 300-400 divided bytotal daily insulin dose in type 1 diabetes patients who use the insulin pump. Diabetes Technol Ther 2012;
14:1077-1080.
6. Nakamura T, Hirota Y, Hashimoto N, et al. Diurnal variation of carbohydrate insulin ratio in adult type 1
diabetic patients treated with continuous subcutaneous insulin infusion. J Diabetes Investig 2014;5:48-50.
7. Davidson PC, Hebblewhite HR, Steed RD, et al. Analysis of guidelines for basal-bolus insulin dosing: basal
insulin, correction factor, and carbohydrate-to-insulin ratio. Endocr Pract 2008;14:1095-1101.
8. Kuroda A, Kaneto H, Yasuda T, et al. Basal insulin requirement is ~30% of the total daily insulin dose in type1 diabetic patients who use the insulin pump. Diabetes Care 2011;34:1089-1090.
9. King AB, Armstrong DU. A prospective evaluation of insulin dosing recommendations in patients with type 1
diabetes at near normal glucose control: basal dosing. J Diabetes Sci Technol 2007;1:36-41.
10. de Bock M, Cooper M, Retterath A, et al. Continuous glucose monitoring adherence: lessons from a clinical- 39 -

Hotta et al
trial to predict outpatient behavior. J Diabetes Sci Technol 2016;10:627-632.
11. Kawasaki E, Maruyama T, Imagawa A, et al. Diagnostic criteria for acute-onset type 1 diabetes mellitus (2012):
Report of the Committee of Japan Diabetes Society on the Research of Fulminant and Acute-onset Type 1
Diabetes Mellitus. J Diabetes Investig 2014;5:115-118.
12. Urakami T, Suzuki J, Yoshida A, et al. Association between sex, age, insulin regimens and glycemic control inchildren and adolescents with type 1 diabetes. Clin Pediatr Endocrinol 2010;19:1-6.
13. Pinhas-Hamiel O, Hamiel U, Boyko V, et al. Trajectories of HbA1c levels in children and youth with type 1
diabetes. PLoS One 2014;9:e109109.
14. Lau YN, Korula S, Chan AK, et al. Analysis of insulin pump settings in children and adolescents with type 1
diabetes mellitus. Pediatr Diabetes 2016;17:319-326.
15. Danne T, Battelino T, Kordonouri O, et al. A cross-sectional international survey of continuous subcutaneous
insulin infusion in 377 children and adolescents with type 1 diabetes mellitus from 10 countries. Pediatr
Diabetes 2005;6:193-198.
16. Walsh J, Roberts R, Bailey T, et al. Guidelines for insulin dosing in continuous subcutaneous insulin infusion
using new formulas from a retrospective study of individuals with optimal glucose levels. J Diabetes Sci
Technol 2010;4:1174-1181.
17. King AB. Continuous glucose monitoring-guided insulin dosing in pump-treated patients with type 1 diabetes:a clinical guide. J Diabetes Sci Technol 2012;6:191-203.
18. Hashimoto T, Kawamura T, Kashihara Y, et al. Factors associated with basal insulin dose in Japanesechildren and young adult type 1 diabetics. J Diabetes Investig 2012;3:276-282.
19. Conrad SC, McGrath MT, Gitelman SE, et al. Transition from multiple daily injections to continuous
subcutaneous insulin infusion in type 1 diabetes mellitus. J Pediatr 2002;140:235-240.
20. Nicolajsen T, Samuelsson A, Hanas R, et al. Insulin doses before and one year after pump start: children havea reversed dawn phenomenon. J Diabetes Sci Technol 2012;6:589-594.
21. Weitzman ED. Biologic rhythms and hormone secretion patterns. Hosp Pract 1976;11:79-86.
22. Rabbone I, Bobbio A, Berger K, et al. Age-related differences in metabolic response to continuous subcutaneous
insulin infusion in pre-pubertal and pubertal children with Type 1 diabetes mellitus. J Endocrinol Invest 2007;
30:477-483.
23. Rose SR, Municchi G, Barnes KM, et al. Spontaneous growth hormone secretion increases during puberty innormal girls and boys. J Clin Endocrinol Metab 1991;73:428-435.
24. Weitzman ED, Fukushima D, Nogeire C, et al. Twenty-four hour pattern of the episodic secretion of cortisol innormal subjects. J Clin Endocrinol Metab 1971;33:14-22.
25. German Nutrition Society (DGE). New Reference Values for Energy Intake. Ann Nutr Metab 2015;66:219-223.

- 40 -

Osaka City Med. J. Vol. 65, 41-54, 2019

Clinical Aspects and Genetic Analysis of Pediatric
Neurofibromatosis Type 1 with Neurological Complications
KENJI FUJITA1), KANAKO YAMASHITA1), TAKAO HOSHINA1), NORIKATSU HIKITA1), TARO SHIMONO2),KAZUYOSHI FUKAI3,4), TOSHIKI TAKENOUCHI5), TOMOKO UEHARA6), KENJIRO KOSAKI6), HIDEYUKI SAYA7),
TAKASHI HAMAZAKI1), and TOSHIYUKI SETO1)
Departments of Pediatrics1), Diagnostic and Interventional Radiology2), andDermatology3), Osaka City University Graduate School of Medicine;
Department of Dermatology4), Osaka City General Hospital; andDepartments of Pediatrics5), Center for Medical Genetics6), andInstitute for Advanced Medical Research7), Keio University School of Medicine

Abstract
Background
Neurofibromatosis type 1 (NF1) is an autosomal dominant disease causing multiple café-au-lait
spots and various systemic signs. NF1 patients often need to be examined complications because oftumorigenesis. Here, with a focus on neurological complications, we studied the clinical features ofthe disease in patients in the Department of Pediatrics, Osaka City University Hospital, Japan, andthe relationship between these features and patient genotype.
Methods
The subjects were 81 patients aged 0 to 44 (average 11.3, median 10.3) years who had visited ourdepartment; they were evaluated retrospectively by examination of their electronic medical records.
We examined family history and characteristic physical findings of NF1, and we assessed patientdevelopment and brain and spine MRIs. In six patients with moderate to severe neurologicalcomplications we also analyzed the NF1 gene.
Results
A total of 45 patients were categorized as having definite NF1 and 24 patients as having suspected
NF1, according to National Institutes of Health criteria. 56 patients had neurological complications
and 12 had optic glioma, pilocytic astrocytoma, schwannoma, or severe plexiform neurofibroma. In
the six patients subjected to genetic analysis we found pathogenic mutations in the NF1, one of which
was undefined. Novel mutations were identified in four patients in three families.
Conclusions
We conclude that NF1 patients should be examined by MRI and genetic analysis during follow-up;

Received September 3 2018; accepted November 27, 2018.
Correspondence to: Toshiyuki Seto, MD, PhD.
Department of Pediatrics, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-3816; Fax: +81-6-6636-8737
E-mail: setot@med.osaka-cu.ac.jp- 41 -

Fujita et al

this is especially true for those with suspected moderate to severe neurological complications. We
need to accumulate more genotypic information on Japanese patients with NF1 to elucidate thegenotype-phenotype relationship.
Key Words: Genetic testing; Mutation; Neurofibromatosis 1; Optic nerve glioma;
Pediatric

Introduction
Neurofibromatosis type 1 (NF1) is a genetic disease that is inherited in an autosomal dominantpattern1-4) and has a reported birth incidence of 1:2000-30001,2,5). The initial signs are usually multiple
café-au-lait spots (mCaLs). Most patients are referred from domestic public health centers to our
Department of Pediatrics or Department of Dermatology at Osaka City University Hospital, Japan,
because of the discovery of mCaLs during infancy. Other signs can be skin-fold freckling,
neurofibroma, Lisch nodules, scoliosis, enlargement of the subdural cavity, optic glioma, low-grade
astrocytoma, pilocytic astrocytoma, plexiform neurofibroma, intellectual disability, andneurodevelopmental disorders 1,2,6). A clinical diagnosis of NF1 is usually arrived at by applying
National Institutes of Health (NIH) criteria (Table 1)7). However, obvious neurofibroma, skin-foldfreckling, and scoliosis usually become apparent after puberty, so the existence of characteristic
neurological signs is important for NF1 diagnosis in some children before the diagnosis can be
confirmed according to NIH criteria. Almost half of all NF1 patients have no family history because
their mutations have arisen de novo6,8,9), so in young children with a suspected diagnosis of NF1 it isimportant to look for neurological signs such as developmental delay. We propose that patients andtheir parents should undergo neuroradiological studies and other examinations at presentation, but
because infants and young children can be difficult to evaluate they may need to be admitted tohospital to receive such studies and further follow-up studies under sedation. Ophthalmologic
examination is also difficult without complete sedation.
Eventually, neurological signs in childhood become increasingly important from the perspective of
not only the diagnosis of NF1 but also the necessary follow-up of neurological complications.
Neuroradiological examinations sometimes reveal characteristic findings in the brains of children
with NF1. Unidentified bright objects (UBOs) have been identified as high-intensity lesions in T2-

Table 1. NIH diagnostic criteria for neurofibromatosis type 1
Two or more of the following clinical features must be present
Six or more café-au-lait macules of more than 5 mm greatest diameter in prepubertal individuals, and more than
15 mm in greatest diameter in postpubertal individuals
Two or more neurofibromas of any type, or one plexiform neurofibroma
Freckling in the axillary or inguinal region
Optic glioma
Two or more iris hamartomas (Lisch nodules)
A distinctive bony lesion, such as sphenoid dysplasia, or medullary narrowing and cortical thickening of thelong bone cortex, with or without pseudoarthrosis
A first-degree relative (parent, sibling, or offspring) with NF1 based on the above criteria
NIH, National Institutes of Health; and NF1, neurofibromatosis type 1.

- 42 -

Clinical and Genetic Aspects of Pediatric NF1

weighted images and fluid-attenuated inversion recovery images during brain MRI in children with
NF110,11). Although most UBOs are regarded as benign tumors (hamartomas or low-grade
astrocytomas), rarely, malignant brain tumors are found during the clinical course of the disease10,12).
Optic glioma (also known as optic pathway glioma) is a benign or malignant tumor that can alsoo c c u r i n c h i l d r e n w i t h N F 113-15).

M o r e o v e r, m a l i g n a n t p e r i p h e r a l s c h w a n n o m a s a n dpheochromocytomas can complicate NF116,17). Because of those tumors some patients (and theirparents) must sacrifice time and quality of life for continuing surgery and chemotherapy.
Unfortunately, there are currently few keys to the prognosis, especially in regard to tumorigenesis,of young children with only mCaLs who are suspected of having NF1. The gene associated with thedisease is NF118,19), the phenotype-genotype relationship in NF1 in regard to prognosis and malignant
tumorigenesis has not yet been defined20,21).
Because there have been few reports of the etiology and genetic analysis of NF1 in Japanesechildren22), we considered it important to explore these features in regard to the various complicationsof NF1̶especially neurological complications. Here, with a focus on the key neurological signs inpediatric patients, we retrospectively examined the clinical aspects of all NF1 patients who visited,
and were followed up by, our Department. We also performed genetic analyses of patients with NF1
and multiple complications.

Materials and Methods
Subjects
Eighty-one patients were evaluated between April 2007 and June 2018. All of them had beenreferred to outpatient clinic of Pediatrics, Osaka City University Hospital, for the purpose ofdiagnosis or further examination of NF1. The patients were aged from 0 to 44 years (average 11.3,
median 10.3 years) at the time of the latest visit, and there were 39 males (48.1%) and 42 females(51.9%). The average follow-up period was 6.0 years (median, 5.5 years).
Evaluation of patient histories and neurological complications
All clinical information was obtained from the electronic medical record system of our hospital.
We investigated family history and the characteristic physical findings of NF1, and we assessed
physical and neurological development and brain and spine MRIs. The MRIs were evaluated by aneuropediatrician23,24) and neuroradiologists. Clinical diagnosis was performed by using the NIH
Consensus Development Conference diagnostic criteria (1988 version) (Table 1).
complications were defined here referring to previous reports

6,25)

Neurologicalas abnormal neuroradiologicalfindings (UBOs, optic glioma, pilocytic astrocytoma, plexiform neurofibroma, pheochromocytoma,
schwannoma); epilepsy; intellectual disability (ID); or neurodevelopmental disorder (NDD), including
autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD) and learning
disorder (LD). Here, we further show the clinical courses of six patients with NF1 who manifested
moderate to severe neurological complications (NDD, optic glioma, severe plexiform neurofibroma);
we also analyzed the NF1 genotypes of these patients and related family members.
Genetic analysis
In the six patients in five families who had multiple complications we also analyzed NF1. DNAs
were purified from blood samples; the target region was enriched by using a TruSight One Sequencing
Panel (Illumina®, San Diego, CA)26) that included NF1, and it was sequenced on a Miseq platform(Illumina®, San Diego, CA). Also, we used the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and the- 43 -

Fujita et al

Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php) to search for thepathological significance of mutations reported in the past. Chromosomal microarray testing using
the Agilent 180K microarray (Agilent Technologies ®, Santa Clara, CA) was performed as described
previously27).
Statistical analysis
Statistical analysis was performed by using analysis of variance (ANOVA) and Tukey s test for
multiple pairwise comparisons. Welch s t-test and JMP (version 12) statistical software (SASInstitute Inc.®, Cary, NC).
This study was approved by the institutional review boards of Osaka City University Graduate
School of Medicine (No. 3325 and No. 3326) and Keio University School of Medicine (No. 20110262).
Written informed consent was obtained from all patients. In the case of patients with intellectual
disability, or children, parents or other legally authorized representatives supplied informed consent.

Results
Figure 1 is a flowchart of all subjects included in the study. MCaLs were found in all patients.
They were thus often the chief presenting complain and the catalyst for further examination. A totalof 69 patients were evaluated as having definite (45 patients) or suspected (24 patients) NF1
according to NIH criteria in Table 1. The clinical signs of all patients are summarized in Table 2.
Comparison of the definite and suspected groups revealed that the average age of the definite groupwas significantly higher than that of the suspected one. The average ages of the definite andsuspected NF groups were much higher than those of the mCaLs-only group. The sex ratio did not
differ significantly among groups, although in the definite NF1 group there were twice as manyfemales as males with no neurological complications. In the definite NF1 group, 33 patients had
neurological complications. In the definite NF1 group, 10 of the patients with neurologicalFigure 1. Flowchart of subjects included in this study. NIH, National Institutes of Health.

- 44 -

Clinical and Genetic Aspects of Pediatric NF1Table 2. Clinical profiles of all patients
Definite NF1

Suspected NF1mCals only

n＝24

n＝12

n＝45
＊

＊

Age (years, average±SD)13.9±8.7

10.2±6.7

4.0±2.9

†
＋

−

＋

−

n＝33 (73)n＝12 (27)n＝23 (96)n＝1 (4)14.1±8.7

13.1±9.1

9.8±6.5

19.5

male

17 (38)4 (9)12 (50)1 (4)5female16 (36)8 (18)

11 (46)0 (0)7Positive family history

17 (38)6 (13)

-

-

-

mCaLs

33 (73)

12 (27)23 (96)1 (4)12Freckling

2 (4)

0 (0)

0 (0)

0 (0)

-

Neurofibroma

13 (29)5 (11)---

Plexiform neurofibroma (severe)

2 (4)

2 (4)

-

-

-

UBO

26 (58) †-23 (96)--

Brain tumor

8 (18) †

-

0 (0)

-

-

optic glioma

7 (16)----

pilocytic astrocytoma

1 (2)

-

0 (0)

-

-

schwannoma

1 (2)

-

0 (0)

-

-

Intellectual disability

4 (9)

-

-

-

-

Neurodevelopmental disorder

13 (29) †

-

1 (4)

-

-

Epilepsy0 (0) †

-

2 (8)

-

-

Electroencephalogram abnormality

1 (2)

-

2 (8)

-

-

Ophthalmologic lesions

16 (36)

4 (9)

0 (0)

0 (0)

-

Lisch nodule

11 (24)

4 (9)

-

-

-

ocular fundus abnormality

5 (11)

3 (7)

0 (0)

0 (0)

-

Scoliosis

7 (16)

3 (7)

-

-

-

Short stature ( ＜ −2SD )

6 (13)

3 (7)

0 (0)

1 (4)

-

Macrocephaly ( ＞ ＋2SD )

3 (7)

1 (2)

4 (16)

0-

Neurological complications
Age (years, average±SD)
Sex

Numbers in parentheses are percentages: the denominator is the number of patients with defined NF1 (n ＝45)
or suspected NF1 (n＝24). About ages, data were analyzed using ANOVA. Significant difference. And, multiple
comparisons are performed using wilcoxon's test. ＊ p＜0.05. For the neurological complications with defined NF1 andsuspected NF1, we compaired with χ2-test. †p＜0.05. mCaLs, multiple café-au-lait spots ; and UBO, unidentified
bright object.

- 45 -

Fujita et alcomplications had optic glioma, pilocytic astrocytoma, schwannoma, or severe plexiform
neurofibroma. Pilocytic astrocytoma were found accidentally while following UBO in patient with ID.
We found neurological complications in 81% of the 69 (73% of definite and 96% of suspected NF1
patients). And we found UBOs in 71.3% of our 69 definite and suspected NF1 patients combined.
Exact evaluation of development and intelligence by using the Wechsler Intelligence Scale forChildren or using the Kyoto scale of psychological development was not performed in all of our
subjects. However, 29% of patients with definite NF1 were diagnosed as having as NDD, including
ASD, ADHD, and LD. There were two patients with definite or suspected NF1 and epilepsy (Westsyndrome or juvenile absence epilepsy) (2.9%). The percentage of patients with neurologicalcomplications was smaller in the definite group than in the suspected group.
Six patients were found to have pathogenic mutations in NF1 (Table 3). In the suspected NF1group, all patients except one had UBO as a neurological complication; the remaining patient had
short stature. Two patients in this group had epilepsy.
Presentations of six patients in the definite NF1 group who had tumors
1. Patient (P) 1
Her birth was normal. However, she gradually began to show developmental delay (DD) and was
followed up at a local health center. She was referred to our hospital because of DD and mCaLs at
age 3 years. We diagnosed her as having ID, ASD, and NF1. Optic glioma was found at the age of 4
years, but at the time of writing it had not progressed (Fig. 2a).
2. P2
He was referred to our hospital by a local hospital because of NF1 at age 13 years. His mother,
grandmother, and uncle had mCaLs. He had undergone surgical procedures because of recurrent
cutaneous neurofibroma, and he had abdominal plexiform neurofibroma (Fig. 2b). He did not have ID
and reported having a normal social life.
3. P3
He was born normally but was followed up initially at a local hospital because of mCaLs. At theage of 9 years, he was referred to our hospital because of NDD and NF1. His brain MRI revealed
multiple UBOs. He was diagnosed at 13 years as having LD. He had systemic cutaneous
neurofibroma and received plastic surgery at 29 years.
4. P4
He was referred to our hospital at age 4 months because of mCaLs. He was of short stature anddeveloped increased numbers and size of mCaLs at age 4 years. He was diagnosed as having ADHD
at age 6 years because of hyperactivity and was treated with atomoxetine. The medication was notvery effective, and he is now controlled with osmotic-release oral system (OROS) methylphenidate.
His brain MRI revealed multiple UBOs (Fig. 2c). He has short stature ( ＜−2SD). His mother was
also diagnosed as having NF1.
5. P5 and P6
P5 and P6 (her younger brother) were referred to our hospital at the age of 3 years and 8 months,
respectively. P5 had mCaLs and mild ID. Her mother had noticed a subcutaneous mass upon her(breech) birth. Her MRI revealed a large plexiform neurofibroma (Fig. 2d). P6 s brain MRI revealed
multiple UBOs and an optic glioma (Fig. 2e). It developed further over a few months (Fig. 2f), so we
performed treatment with chemotherapy. Details of all five patients with mutations are summarizedin Table 3.

- 46 -

Clinical and Genetic Aspects of Pediatric NF1Figure 2. MRI findings in selected NF1 patients.
a, Optic glioma in patient 1 (white arrow). She had an optic glioma on the right optic nerve at age 4 years.
b, Abdominal plexiform neurofibroma in patient 2 (white arrows). He was aware of a subcutaneous mass in the rightupper abdomen, and it gradually enlarged.
c, Multiple unidentified bright objects in patient 4 (white arrows). He had no malignant tumors.
d, Large plexiform neurofibroma in patient 5, from the sacrum to the distal femur (white arrows). Her parents had
noticed her subcutaneous mass at the time of breech birth.
e, Optic glioma in the patient 6. An optic glioma (white arrow) was found by follow-up MRI study of unidentified
bright objects.
f, MRI of the same patient 6 months later since Figure 2e revealed the optic glioma developed obviously (white arrow).
T1WI, T1-weighted image; T2WI, T2-weighted image; CE, contrast enhanced; and FLAIR, fluid-attenuated inversion
recovery.

Determination of genotype
Genetic analysis was performed in all of the patients described above. The pathogenic mutation
was defined in all except for P2. P1 had a de novo missense mutation, c.2350T ＞C (p.Trp784Arg).
Although P2 had neither pathogenic mutation in the exons nor microdeletion/duplication upon arraycomparative genomic hybridization analysis, an intron mutation was found. Its pathogenic meaning
remains unknown. P3 had a frameshift mutation, c.3527delG (p.Arg1176Lys fs＊8). P4 and his
mother had a nonsense mutation, c.1603C ＞T (p.Gln535＊). P5 and P6 and mother had a splicing
mutation, c.888 ＋2T ＞A. The mutations in P3, P4, and P5 (P6) were novel. All results are
summarized in Table 3.

- 47 -

- 48 -

F＋＋

borderline−

borderline

mild

mild

NE

normalNE

48＊

moderateID＋−

history

Family

NE

ASD

ASD

NE

ADHD

LD

NE

ASD

NDD

＋

−

＋

−

−

−

＋

NE

＋

Malignant

−

−

NE

−

−

−

−

NE

glioma brain tumor

NE

＋

＋

−

＋

UBO

Optic

NE

＋

NE

＋

NE

＋

NE

Nf

PlexiformNEheadache

HL, vertigo

macrocephaly

funnel chest

Other

NE

−

−

NE

＋

＋

＋

＋

nodules

Lisch

＋

＋

＋

＋

＋

＋

＋

＋

mCaLs

Short

＋

−

＋

＋

＋

＋

＋

−

Nf

−

−

−

＋

＋

−

−

−

subcutaneous stature

and/orCutaneous

NE

−

−

NE

−

＋

＋

−

Scoliosis

20

location

Exon

missenseTypeinformation)(no clinical＋past reports

Relatedc.888＋2T ＞Ac.1603C ＞Tc.3527delG

8

14

27

abnormality

splicing

termination

frameshift(novel)−(novel)−(novel)−(no pathogenic mutation determined)

c.2350T ＞Cmutation

Identified

Results of genetic analysis

In this table, the patient's age is as of june 2018. NF1, neurofibromatosis type 1; ID, intellectual disability. It evaluated by using the Kyoto scale of psychologicaldevelopment or the Wechsler Intelligence Scale for Children; NDD, neurodevelopmental disorder; UBO, unidentified bright object; Nf, neurofibroma; mCaLs, café-au-lait
spots; P, patient; ASD, autism spectrum disorder; NE, not examined; LD, learning disorder; HL, hearing loss; and ADHD, attention-deficit hyperactivity disorder.

mother

P5 and 6's

34

M

3

(P5's brother)

P6

F

6

P5

F

34

M

M

P4's mother

32

P3

M

12

23

P2

F

Sex

P4

5

(years)P1

Patient

Age

Neurological complications

Table 3. Relationships between neurological complications and NF1 genotype in five patients and their family members

Fujita et alClinical and Genetic Aspects of Pediatric NF1Discussion
We retrospectively elucidated the clinical features of all patients presenting to our department
with definite or suspected NF1 during the study period. In this literature, we present two new
findings. First, examining their follow-up MRIs, optic gliomas and pilocytic astrocytoma wereincidentally detected in young children having ID. Second, we identified novel mutations in four
patients with NF1.
In our study, 12 patients (15% of all subjects) were judged clinically in childhood by pediatriciansas having only mCaLs (Table 2). This is because mCaLs are generally the initial chief complaint
prompting referral to our pediatrics or dermatology departments from domestic public health centersor clinics. These patients general condition was good and they showed no more clinical signs during
their follow-up periods. They included children with tiny and mild mCaLs. We needed to decide
whether to diagnose these patients as having suspected NF1 and to follow them up and examine
them for various complications, or to stop our observations and label the mCaLs as mere macules or
nevus syndromes. Distinction between NF1 and Legius syndrome (in which there are mCaLs with orwithout freckling, but never the typical NF1-associated tumors 28)) is possible by genetic analysis.
However, in Japan such analyses are not common. Some doctors in charge of the children with only
mCaLs planned to examine them by MRI, possibly because of certain aspects of their behavior or an
impression that their mCaLs were severe. As our study was not prospective, further reasons for thelost to follow-up were unknown. Most of the doctors in charge of patients with suspected NF1 arechild neurologists in our department. If they were to notice even a tiny objective finding̶such as
behavior suggestive of motor or developmental delay̶they would suggest to a child s parents thatthe child receive further examinations or be observed at an outpatient clinic for a while. NF1 is a
wide-spectrum disorder ranging from very mild to very severe. Moreover, children are often hard toevaluate in some examinations (e.g. MRI) without deep sedation, and some signs tend to appear after
puberty. This is a reason to have no choice to postpone clarifying in childhood whether or not thepatient definitely has NF1.
Next, we discussed neurological complications among 69 NF1 patients̶24 with suspected NF1
and 45 with definite disease̶by retrospective electronic records analysis. In our study, We foundneurological complications in many NF1 patients. One Japanese report found neurologicalcomplications (except for brain tumor) in 38.1% of patients 29). UBO is a characteristic major finding
in the brains of NF1 patients. The reason for this higher percentage compared with the previous
study is because it is child neurologists who mostly see and follow patients with NF1, and young
children suspected of having NF1 commonly have only UBOs plus mCaLs before their diagnoses are
confirmed. The average age of patients with suspected NF1 was lower than that of patients withdefinite NF1. It is regarded as one possibility that the percentage of patients with neurologicalcomplications was smaller in the definite group than in the suspected group because that growing
patients with suspected NF1 likely move into the definite group as they age and begin developing
signs other than UBO.
When examined across several studies, the frequency of LD as an NDD in children with NF1 isestimated to be between 30% and 65%25,30). This value is not significantly different from our study.
The complication rate of epilepsy is also much higher than the percentage of the general populationof Japanese children with epilepsy (0.3% to 0.5%) 31). Another patient showed obvious abnormal
epileptic discharges in her electroencephalograph. Seizures are reported elsewhere to occur in 4% to- 49 -

Fujita et al

9% of patients with NF132). Relative to those in the general population, seizures in people with NF1
are more often focal and related to the presence of brain tumors. Moreover, individuals with NF1 andseizures frequently require more aggressive therapy than those without NF1, and some patients with
NF1-related epilepsy should be considered for surgery when appropriate25). NDD and epilepsy often
go together in NF1, and we need to pay close attention to them̶in the same way as for brain
tumors̶during follow-up, as indicated in previous reports25).
Once NF1 is diagnosed by a dermatologist, pediatrician, or medical geneticist, there is a need tostart surveying for the occurrence of brain tumors and other complications. Therefore, early
diagnosis is important, and we expect that patient genotyping will make it possible to determine eachpatient s prognosis, especially in terms of tumorigenesis. NF1 encodes neurofibromin and is locatedon chromosome 17q11.2. It is 350 kb long and has 60 exons6,19,33,34). There are some reports in whichgene mutations have been associated with phenotypes. First, patients with NF1 microdeletion
usually have more severe complications and are at increased risk of malignant peripheral sheathtumors35). Second, a specific single NF1 amino-acid deletion [e.g. c.2970_2972del (p.Met992del)] could
result in mild complications 36). Third, a missense mutation (p.Arg1809Cys substitution) in
neurofibromin could be a cause of NDD, ID, and optic glioma37,38). Recently, it was reported that theneighboring codons 844 to 848 are involved in severe NF1 phenotypes such as bone abnormalities,
plexiform or symptomatic spinal neurofibroma (or both), symptomatic optic glioma, and othermalignant neoplasms21). Other reports have suggested that NF1 patients with mutations located in
the cysteine- and serine-rich domain (CSRD) were at higher risk of optic glioma than patients with
mutations in other regions39). In mice, NF1-patient-derived germline NF1 mutations (c.2542G ＞Cand C.2041C ＞T) are reportedly associated with optic glioma formation40). It is also known that,generally, microdeletion, truncation, or frameshift mutations tend to be phenotypically severe 41).
However, according to previous reports, an obvious genotype-phenotype relationship has not yet been
elucidated6,21).
We presented details of six patients with NF1. All patients had both NDD and UBO except for P2,
with or without moderate to severe plexiform neurofibroma or optic glioma. We performed a nextgeneration sequencing analysis of these patients with NF1 and relatively severe neurologicalcomplications after genetic counseling of the children and their parents by a medical geneticist.
Eventually, we identified four kinds of pathogenic mutations in NF1, including three novel mutations.
P1 had optic glioma, moderate ID, and ASD. The mutation identified in P1 was previously reported
as pathogenic42), but there was no clinical information in that previous report. This mutation is
located in the CSRD domain reported of relation with optic glioma39). A mutation was found in an
intron of NF1 in P2, but its pathogenic significance could not be determined. P3 had LD, chronicheadache, and severe systemic subcutaneous neurofibroma; a frameshift was identified in exon 27
between the tubulin-binding domains (TBD) and GAP-related domains (GRD), which interact withRas protein (Fig. 3). The novel termination mutation identified in P4 and his mother resulted in
short stature in both. P4 has severe UBO and ADHD, but his mother has not been examined. The
mutation identified in P5 s family shows interfamilial phenotypic variability. This may have been
due to clinical variability or to differences in transcripts in terms of splicing mutations, differences inpenetration rates or epigenetic factors, or other reasons.
We have compared our identified mutations with those described in reports mentioned above (Fig.
3). Neurofibromin encoded by NF1 regulates the activity of Ras protein by inactivating it through- 50 -

Clinical and Genetic Aspects of Pediatric NF1Figure 3. Scheme of NF1 gene and neurofibromin protein. This scheme is based on the figure in references 39 and
44. NF1 contains 60 exons, including three alternatively spliced exons. Mutation in the CSRD domain, which interacts
with RAS protein, carries a higher risk of optic glioma than mutations in other regions. SM, splicing mutation; TM,
termination mutation; MM, missense mutation; FS, frameshift mutation; CSRD, cysteine- and serine-rich domain;
TBD, tubulin binding domain; GRD, domain responsible for interaction with and GTP hydrolysis; SEC-PH, bipartite
lipid-binding domain; FAK, focal adhesion kinase-interacting domain; and NLS, nuclear localization sequence.

dephosphorylation 43) . In other words, it negatively regulates the activity of Ras protein;neurofibromin is a GTP-ase activating protein that accelerates the hydrolysis of Ras-bound GTP43,44).
Mutations in the CSRD domain interacting with Ras protein carry a high risk of optic glioma39). Optic
glioma is the most common brain tumor associated with NF1, with as many as 15% or 20% of children
with NF1 harboring an optic pathway tumor15,45). The lifetime incidence of brainstem gliomas in NF1
is 4%, with presentation typically before the age of 10 years46).
NF1 carries a high risk of tumorigenesis because it is a RASopathy. Therefore, we recommend
examination by MRI, avoiding radiological CT scans, especially in childhood. CT is too low resolution
to evaluate UBOs and other small tumors, and repeated follow-up examination by this method is notrecommended because of the need to minimize radiation exposure in children that are already at riskof tumorigenesis. However, it is still controversial to examine routine screening of MRI for early
identification of optic glioma in patients with NF1 in recent reports6,18,30,47,48). We need to performsemiological and behavioral observations in NDD, examine electroencephalograms in epilepsy, andperform careful systemic physical examinations̶especially by palpation around the abdomen andback. Along with these evaluations of the various complications of NF1, we can consider genetic
analysis of the patients in cooperation with medical geneticists and genetic counselors. To elucidate
phenotype-genotype relationships in NF1, we need to accumulate gene analysis data and conduct
further studies with appropriate and accurate genetic counseling. In this study our research was
limited by its retrospective nature.
A larger study in Japan is required in the future to predict accurately the prognosis of thesepatients, especially regarding the development of malignant optic glioma and other tumors. We
conclude that NF1 patients should be examined by MRI and genetic analysis during follow-up; this isespecially true for those with suspected moderate to severe neurological complications. We need to- 51 -

Fujita et alaccumulate more genotypic information on Japanese patients with NF1 to elucidate the genotypephenotype relationship.
Acknowledgments
All authors have no COI to declare regarding the present study.
We thank Emeritus Prof. Haruo Shintaku, Dr. Satoru Sakuma, Dr. Tomoyuki Kawamura, and Dr.
Sadao Tokimasa from our Department of Pediatrics; Clinical Prof. Hiroaki Sakamoto and Dr.
Takehiro Uda from our Department of Neurosurgery, who followed up some of the patients with NF1.
We also thank Ms. Yukiko Okayama, clinical technologist in the Clinical Laboratory at Osaka City
University Hospital; Ms. Tomoko Sakaguchi and Ms. Noriko Nakano, technical assistants in the
Department of Pediatrics; and members of the research support platform at Osaka City University
Graduate School of Medicine.
This study was supported by research funds of program for integrated database of clinical andgenomic information from the Japan Agency for Medical Research and Development(JP18kk0205002).
Kanako Yamashita, and Toshiyuki Seto mainly followed the patients and wrote the first draft ofthis manuscript. No honorarium, grant, or other form of payment was received to produce this paper.
Takao Hoshina and Norikatsu Hikita performed various examinations of NF1 and helped with thestudy. Takashi Hamazaki helped with the study and gave advice. Taro Shimono mainly evaluated
MRI studies of the patients as a neuroradiologist. Kazuyoshi Fukai mainly followed the patients as adermatologist. Toshiki Takenouchi, Tomoko Uehara, and Kenjiro Kosaki performed genetic analyses
and gave advice on the study. Hideyuki Saya gave us the opportunity to start the study. Toshiyuki
Seto designed and supervised all the research.
Part of this report was presented by K. Yamashita et al at the 7th annual meeting of the JapaneseSociety of Recklinghausen Disease, Tokyo, Japan, on 29 November 2015. Another part of this study
was presented by K. Fujita et al at the annual meeting of the American Society of Human Genetics,
Orlando, FL, USA, from 17 to 21 October 2017.
References
1. Sahin M. Neurofibromatosis. In: Kliegman R, Stanton BF, St Geme JW, Schor NF, Behrman RE, Nelson WE,
editors. Nelson textbook of pediatrics.20 ed. Philadelphia: Elsevier/Saunders; 2016. pp. 2874-2877.
2. Thiele EA, Korf BR. Phakomatoses and Allied Conditions. In: Swaiman KF, Ashwal S, Ferrieo DM et al,editors. Pediatric neurology. 5 ed. Philadelphia: Elsevier/Saunders; 2012. pp. 497-502.
3. Maria BL, Menkes JH. Neurocutaneous syndromes. In: Menkes JH, Sarnat HB, Maria BL, editors. Child
Neurology. 7 ed.: Lippincott Williams & Willkins; 2005. pp. 803-809.
4. Hamosh A, McInnes RR, Nussbaum RL, et al. Clinical Case Studies Illustrating Genetic Principle. In :Nussbaum RL, McInnes RR, Willard HF. editors. Thompson & Thompson Genetics in Medicine E-Book. 8 ed:
Elsevier Health Sciences; 2015. pp. 993-996.
5. Lammert M, Friedman JM, Kluwe L, et al. Prevalence of neurofibromatosis 1 in German children atelementary school enrollment. Arch Dermatol 2005;141:71-74.
6. Gutmann DH, Ferner RE, Listernick RH, et al. Neurofibromatosis type 1. Nat Rev Dis Primers 2017;3:17004.
7. Neurofibromatosis. Conference statement. National Institutes of Health Consensus Development Conference.
Arch Neurol 1988;45:575-578.
8. Ars E, Kruyer H, Morell M, et al. Recurrent mutations in the NF1 gene are common among neurofibromatosis
type 1 patients. J Med Genet 2003;40:e82.
9. Zhang J, Tong H, Fu X, et al. Molecular characterization of NF1 and neurofibromatosis type 1 genotypephenotype correlations in a Chinese population. Sci Rep 2015;5:11291.
10. Vezina G, Barkovich AJ. The Phakomatoses. In:Barkovich AJ, Raybaud C. editors. Pediatric Neuroimaging. 5- 52 -

Clinical and Genetic Aspects of Pediatric NF1
ed. Lippincott Williams & Willkins; 2012. pp. 569-588.
11. Sellmer L, Farschtschi S, Marangoni M, et al. Non-optic glioma in adults and children with neurofibromatosis 1.
Orphanet J Rare Dis 2017;12:34.
12. Rodriguez FJ, Perry A, Gutmann DH, et al. Gliomas in neurofibromatosis type 1: a clinicopathologic study of
100 patients. J Neuropathol Exp Neurol 2008;67:240-249.
13. Listernick R, Charrow J, Greenwald MJ, et al. Optic gliomas in children with neurofibromatosis type 1. J
pediatr 1989;114:788-792.
14. Listernick R, Louis DN, Packer RJ, et al. Optic pathway gliomas in children with neurofibromatosis 1:
consensus statement from the NF1 Optic Pathway Glioma Task Force. Ann Neurol 1997;41:143-149.
15. Lewis RA, Gerson LP, Axelson KA, et al. von Recklinghausen Neurofibromatosis: Ⅱ. Incidence of optic
gliomata. Ophthalmology 1984;91:929-935.
16. Evans DG, Baser ME, McGaughran J, et al. Malignant peripheral nerve sheath tumours in neurofibromatosis
1. J Med Genet 2002;39:311-314.
17. Opocher G, Conton P, Schiavi F, et al. Pheochromocytoma in von Hippel-Lindau disease and neurofibromatosis
type 1. Fam Cancer 2005;4:13-16.
18. Ferner RE, Huson SM, Thomas N, et al. Guidelines for the diagnosis and management of individuals withneurofibromatosis 1. J Med Genet 2007;44:81-88.
19. Wallace MR, Marchuk DA, Andersen LB, et al. Type 1 neurofibromatosis gene: identification of a largetranscript disrupted in three NF1 patients. Science 1990;249:181-186.
20. Bianchessi D, Morosini S, Saletti V, et al. 126 novel mutations in Italian patients with neurofibromatosis type 1.
Mol Genet Genomic Med 2015;3:513-525.
21. Koczkowska M, Chen Y, Callens T, et al. Genotype-phenotype correlation in NF1: evidence for a more severe
phenotype associated with missense mutations affecting NF1 codons 844-848. Am J Hum Genet 2018;102:6987.
22. Hirabaru K, Matsuo M. Neurological comorbidity in children with neurofibromatosis type 1. Pediatr Int 2018;60:70-75.
23. Seto T, Kono K, Morimoto K, et al. Brain magnetic resonance imaging in 23 patients withmucopolysaccharidoses and the effect of bone marrow transplantation. Ann Neurol 2001;50:79-92.
24. Seto T, Takesada H, Matsushita N, et al. Twelve-year-old girl with intracranial epidural abscess andsphenoiditis. Brain Dev 2014;36:359-361.
25. Anderson JL, Gutmann DH. Chapter 4 - Neurofibromatosis type 1. In: Islam MP, Roach ES, editors. Handbookof Clinical Neurology: Elsevier; 2015. pp. 75-86.
26. Maruoka R, Takenouchi T, Torii C, et al. The use of next-generation sequencing in molecular diagnosis ofneurofibromatosis type 1: a validation study. Genet Test Mol Biomarkers 2014;18:722-735.
27. Yagihashi T, Torii C, Takahashi R, et al. Clinical utility of an array comparative genomic hybridization
analysis for Williams syndrome. Congenit Anom (Kyoto) 2014;54:225-227.
28. Messiaen L, Yao S, Brems H, et al. Clinical and mutational spectrum of neurofibromatosis type 1–like
syndrome. JAMA 2009;302:2111-2118.
29. Yoshida Y, Ehara Y, Koga M, et al. Epidemiological analysis of major complications requiring medical
intervention in patients with neurofibromatosis 1. Acta Derm Venereol 2018;98:753-756.
30. North K, Riccardi V, Samango-Sprouse C, et al. Cognitive function and academic performance inneurofibromatosis. 1: consensus statement from the NF1 Cognitive Disorders Task Force. Neurology 1997;
48:1121-1127.
31. Kulkantrakorn K, Geller TJ. Seizures in neurofibromatosis 1. Pediatr Neurol 1998;19:347-350.
32. Camfield P, Camfield C. Incidence, prevalence and aetiology of seizures and epilepsy in children. Epileptic
Disord 2015;17:117-123.
33. Ballester R, Marchuk D, Boguski M, et al. The NF1 locus encodes a protein functionally related to mammalian
GAP and yeast IRA proteins. Cell 1990;63:851-859.
34. Cawthon RM, Weiss R, Xu G, et al. A major segment of the neurofibromatosis type 1 gene: cDNA sequence,
genomic structure, and point mutations. Cell 1990;62:193-201.
35. Kehrer-Sawatzki H, Mautner VF, Cooper DN. Emerging genotype-phenotype relationships in patients withlarge NF1 deletions. Hum Genet 2017;136:349-376.
36. Upadhyaya M, Huson SM, Davies M, et al. An absence of cutaneous neurofibromas associated with a 3-bp
inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1
genotype-phenotype correlation. Am J Hum Genet 2007;80:140-151.
37. Pinna V, Lanari V, Daniele P, et al. p.Arg1809Cys substitution in neurofibromin is associated with a distinctive
NF1 phenotype without neurofibromas. Eur J Hum Genet 2015;23:1068-1071.

- 53 -

Fujita et al
38. Rojnueangnit K, Xie J, Gomes A, et al. High incidence of noonan syndrome features including short statureand pulmonic stenosis in patients carrying NF1 missense mutations affecting p.Arg1809: genotype-phenotype
correlation. Hum Mutat 2015;36:1052-1063.
39. Xu M, Xiong H, Han Y, et al. Identification of mutation regions on NF1 responsible for high- and low-risk
development of optic pathway glioma in neurofibromatosis typeⅠ. Front Genet 2018;9:270.
40. Toonen JA, Anastasaki C, Smithson LJ, et al. NF1 germline mutation differentially dictates optic glioma
formation and growth in neurofibromatosis-1. Hum Mol Genet 2016;25:1703-1713.
41. Hamosh A, McInnes RR, Nussbaum RL, et al. Types of mutations and their consequences. In : Nussbaum RL,
McInnes RR, Willard HF. editors. Thompson & Thompson Genetics in Medicine E-Book. 8 ed: Elsevier Health
Sciences; 2015. pp. 125-128.
42. Kluwe L, Friedrich RE, Korf B, et al. NF1 mutations in neurofibromatosis 1 patients with plexiform
neurofibromas. Hum Mutat 2002;19:309.
43. Ratner N, Miller SJ. A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour
suppressor. Nat Rev Cancer 2015;15:290-301.
44. Abramowicz A, Gos M. Neurofibromin in neurofibromatosis type 1 - mutations in NF1 gene as a cause of
disease. Dev Period Med 2014;18:297-306.
45. Listernick R, Charrow J, Greenwald MJ, et al. Optic gliomas in children with neurofibromatosis type 1. J
Pediatr 1989;114:788-792.
46. Molloy PT, Bilaniuk LT, Vaughan SN, et al. Brainstem tumors in patients with neurofibromatosis type 1. adistinct clinical entity. Neurology 1995;45:1897-1902.
47. Listernick R, Charrow J, Greenwald M. Emergence of optic pathway gliomas in children withneurofibromatosis type 1 after normal neuroimaging results. J pediatr 1992;121:584-587.
48. King A, Listernick R, Charrow J, et al. Optic pathway gliomas in neurofibromatosis type 1: the effect ofpresenting symptoms on outcome. Am J Med Genet A. 2003;122A:95-99.

- 54 -

Osaka City Med. J. Vol. 65, 55-63, 2019

Change in the Thickness of Retinal Layers after Selective Retina
Therapy (SRT) in Patients with Central Serous Chorioretinopathy
KUMIKO HIRAYAMA1), MANABU YAMAMOTO1), TAKEYA KOHNO1), DIRK THEISEN-KUNDE2),
RALF BRINKMANN2,3), YOKO MIURA2-4), and SHIGERU HONDA1)
Department of Ophthalmology and Visual Science1), Osaka City University
Graduate School of Medicine; Medical Laser Center Luebeck2); andInstitute of Biomedical Optics3) and Department ofOphthalmology4), University of Luebeck

Abstract
BackgroundTo investigate retinal thickness after selective retina therapy (SRT) for central serous
chorioretinopathy (CSC) and the factors related to visual prognosis.
Methods
This retrospective study examined 23 eyes of 21 consecutive patients (17 males, 4 females) with
CSC and followed-up for ＞6 months. A SRT (Q-switched Nd:YLF laser, 527 μm, pulse duration 1.7µs, 30 pulses per irradiation spot with a frequency of 100 Hz) was used for the treatment. The needfor re-treatment was judged every three months after the initial treatment. The best corrected visualacuity (BCVA) and central macular thickness (CMT) were compared among pre-treatment, at thetime of resolution, and at final follow-up examination. Furthermore, the inner retinal thickness (IRT)and outer retinal thickness (ORT) at the time of resolution and at final follow-up, and their rates ofchange were also investigated.
Results
Final BCVA improved (logMAR: from 0.01±0.22 to −0.10±0.15) and the CMT decreased (from336±78 µm to 185±28 µm) significantly after SRT compared with pre-treatment. The IRT did notchange from the time at resolution to the final examination, while the ORT increased significantly
during this time period (from 77±11 µm to 86±10 µm). A stepwise regression analysis identified thatthe pre-treatment BCVA and the rate of ORT increase have a significant positive correlation with thefinal BCVA.
Conclusions
SRT may improve final visual acuity of the patient of CSC, which is accompanied by the increasein the thickness of the outer retinal layer shortly after the resolution of subretinal fluid.

Received September 12, 2018; accepted November 27, 2018.
Correspondence to: Manabu Yamamoto, MD.
Department of Ophthalmology and Visual Sciences, Osaka City University Graduate School of Medicine,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan
Tel: +81-6-6645-3867; Fax: +81-6-6645-3873
E-mail: manabun@msic.med.osaka-cu.ac.jp- 55 -

Hirayama et al

Key Words: ‌Laser therapy; Central serous chorioretinopathy; Retina; Subretinal fluid

Introduction
Central serous chorioretinopathy (CSC) is accompanied by subretinal fluid (SRF) of the macularregion, and can lead to reduced visual acuity, metamorphopsia, micropsia, central scotoma, anddecreased contrast sensitivity, etc 1,2). It is a disease that can be expected to resolve spontaneously
within a few months, with a good prognosis for visual acuity. However, there are some cases in which
SRF is long-standing, which often develop irreversible damage to the neurosensory retina and retinal
pigment epithelium (RPE), sometimes with permanent sequelae3-5).
Selective retina therapy (SRT) was developed as a laser procedure in which the RPE cells are
selectively disrupted without affecting the neural retina or choroid 6-8). In this procedure, amicrosecond pulsed laser is used to induce an instantaneous increase in the temperature of just at
the melanosomes in the RPE. When exceeding the vaporization temperature microbubbles aroundthe melanosomes occur, resulting in a temporary expansion of the volume of the cells that causes
mechanical disruption of the RPE cells. Due to the spatial and temporal thermal confinement
through the laser pulse shorter than the thermal relaxation time of the RPE, there is no significant
temperature increase in the surrounding tissues which might cause thermal damage9,10).
SRT is known to be effective for CSC, diabetic macular edema (DME), and prolonged SRF after
retinal detachment11-17). The therapeutic mechanism of SRT is to disrupt diseased RPE cells andpromote the regrowth of the surrounding healthy ones. We previously reported the safety of SRT for
CSC using microperimetry up to 3 months after treatment 17). Although it is known that SRT is
effective in the short term to resolve subretinal fluid for CSC, there has been no study on the factors
related to visual acuity after SRT. The current study aimed to observe the retinal thickness after
SRT in detail and to investigate factors related to visual prognosis.

Methods
This retrospective study examined 23 eyes of 21 consecutive patients (17 males, 4 females)
diagnosed with CSC and followed-up for more than 6 months at Osaka City University Hospitalduring the study period from July 2011 to July 2013. The average patient age was 48.1 years (range,
29 to 67 years). The basic clinical data of all patients are shown in Table 1.
The inclusion criteria were as follows: (1) minimum age of 20 years; (2) subjective symptoms ofcentral scotoma, metamorphopsia, or decline of visual acuity; (3) history of more than 3 months withno sign of improvement of CSC [diagnosed with optical coherence tomography (OCT)]; (4) presence of
SRF on OCT; (5) presence of active leakage on fluorescence angiography (FA); and (6) SRF
disappeared within 1 year after the last SRT, and no recurrence occurred for more than 3 months
after resolution of SRF.
The exclusion criteria were as follows: (1) having another retinal disease; (2) having a condition
associated with choroidal neovascularization; (3) prior history of another therapy such as PDT or
local retinal photocoagulation within the past 6 months; (4) having a systemic inflammatory disease;(5) haemorrhagic diathesis or taking anticoagulants; (6) having a systemic disease such as untreatedhypertension or diabetes mellitus; or (7) pregnant or possibly pregnant.
SRT laserThis study was approved by the Institutional Review Board (IRB) of the University hospital (No.

- 56 -

Thickness of Retinal Layers after SRT
Table 1. Patient characteristics before treatment
Characteristics
Number of patients

21 Cases (23 eyes)

Sex (Cases; eyes)

Male 17; 19, Female 4; 4Age; Mean (Range)

48.1 (29-67)

Number of episodes (%)
First

13 (56.5)

Second or more

10 (43.5)

Characteristics of leakage in FA (%)
Focal leakage

18 (78.3)

Diffuse leakage

5 (21.7)

Duration of symptom (months); Median (Range)

6 (3-24)Pretreatment BCVA (logMAR); Mean (Range)

0.03 (−0.30-0.70)

Pretreatment CMT (μm); Mean (Range)

336.0 (226-475)

FA, fluorescein angiography; BCVA, best corrected visual acuity; logMAR, logarithm of the minimum
angle of resolution; and CMT, central macular thickness.

2009 and 2421), based on the Declaration of Helsinki, and was registered with University Hospital
Medical Information Network (UMIN) (No. 000005396). Written informed consent was acquired fromall patients. The SRT laser system used was developed by the Medical Laser Center Lübeck (Lübeck,Germany). The laser itself is a Q-switched Nd:YLF-laser (wavelength 527 nm) with a pulse durationof 1.7 μs, which applies 30 pulses per irradiation spot with a frequency of 100 Hz. A Mainster central
field contact lens with a magnifying power of 1.05 was used and adjusted so that the spot size on theretina is 200 μm.
The induced thermomechanical cell damage is not ophthalmoscopically visible during SRT,
therefore the generation of microbubbles in the RPE cells and the subsequent RPE cell destruction
were inferred from the ultrasonic wave being induced from the microbubble expansion. Theseultrasonic waves were measured with a transducer embedded in the contact lens. This signal wasprocessed by a software algorithm to compute an optoacoustic value (OA value) as reported earlier by
Yasui et al 17), which serves as an indicator for microbubble formation and thus cell disruption.
After performing visual acuity testing, slit-lamp microscopy, OCT, and fundus autofluorescence,
test irradiations were conducted around vascular arcades upper or lower part of the macula in orderto decide the range of treatment laser energy. The test irradiation was performed with the energy
started at 60 μJ, followed by increases with 20-μJ increments, until the OA value reached several
hundred. After test irradiations, FA and Indocyanine Green Angiography (IA) were performed, andleakage from the test irradiation sites was confirmed. The main laser treatment was initiated withthe lowest energy level at which leakage was observed in test irradiation. If the OA value is low
during treatment, the treatment energy was adjusted such that the OA value might be aroundseveral hundred. Following the main treatment, FA was repeated, and after confirming the increaseof fluorescein leaks from the treated region, the treatment was concluded.
The need for re-treatment was judged every three months after the initial treatment. If OCT
showed residual SRF in the macular region, FA was conducted, and the presence or absence of- 57 -

Hirayama et al

fluorescein leaks was confirmed. If fluorescein leaks were present, the leakage site was irradiatedwith intervals of 2 to 3 spots diameter, whereas the SRF region was diffusely irradiated in the samemanner if no clear leakage points were observed.
Clinical examinations
Best corrected visual acuity (BCVA) and central macular thickness (CMT) were compared among
three points in time; before treatment, at the time of resolution, and at the final follow-up
examination. The resolution time was defined as the complete disappearance of the SRF on OCT.
The time point of the final follow-up examination was at least 3 months after SRF resolution. As for
BCVA, decimal visual acuity was measured and converted to logarithm of the minimum angle ofresolution (logMAR) for analysis. CMT, defined as the thickness of the retina in the central area, wasmeasured by OCT using a Heidelberg SPECTRALIS® (Heidelberg Engineering GmbH, Heidelberg,
Germany). On the OCT images from the time of resolution and final follow-up, the installed image
processing software was used to carry out intraretinal segmentation by dividing the retina into twolayers, an inner layer and an outer layer, with the external limiting membrane as the dividing line,
and their thickness were then compared. The associations between final BCVA and sex, age,recurrent or de novo CSC, time period of subjective symptoms, time required for resolution, visualacuity, and CMT as pretreatment factors, and with inner retinal thickness (IRT) and outer retinal
thickness (ORT) at the time of resolution and at final follow-up and their rates of change as posttreatment factors were investigated (Fig. 1). Factors for which p＜0.20 on single correlation analysis
were entered into multivariate analysis by stepwise regression analysis.
Statistical analysis
Differences in BCVA were tested using Wilcoxon's signed-rank test for significance, and differences

Figure 1. A horizontal scan of spectral domain optic coherence tomography (SD-OCT) through the central fovea.
Central macular thickness (CMT) is defined as the distance between the surface of the inner limiting membrane (ILM)
and the inner border of the Bruch s membrane (BM) at the central fovea. Inner retinal thickness (IRT) is measured as
the distance between the outer border of the ILM and the inner border of the external limiting membrane (ELM). IRT
is almost the same as outer nuclear layer (ONL) at fovea. Outer retinal thickness (ORT) is measured as the distance
between the outer border of the ELM and the inner border of the BM.

- 58 -

Thickness of Retinal Layers after SRT

in CMT, IRT, and ORT were tested using a paired t-test. Correlations were investigated using
Pearson's correlation coefficient for pairs of normally distributed variables and Spearman's rankcorrelation coefficient for non-normally distributed variables. Associations with sex and recurrence
status were tested using the Mann-Whitney U test. When comparing three groups, a paired t-test
was performed for every pair of group, and the p values were adjusted using the False Discovery Rate
method. The statistical software used was IBM® SPSS® Statistics 24.0 (IBM Japan, Ltd., Tokyo,
Japan), and p＜0.05 was regarded as significant.

Results
The number of test irradiations required for initial treatment ranged from 4-12 (mean 7.8), andthe number of irradiations required for the main laser treatment ranged from 2-17 (mean 7.0). The
irradiation energy used in initial treatment ranged from 62-184 µJ (median 116 µJ), and the OA value
was 54-6455 (median 225). The number of SRT sessions required until SRF resolution was 1-3 (mean1.3). The time from initial treatment to resolution was 1-12 months (median 1 month), and the timefrom resolution to final follow-up was 3-6 months (median 4 months). A representative case of CSC isshown in Figure 2.
Mean BCVA (logMAR) was 0.01±0.22 pre-treatment, −0.04±0.20 at the time of resolution, and−0.10±0.15 at final follow-up, with significant differences between the values at final follow-up andFigure 2. A representative case of central serous chorioretinopathy (CSC). Horizontal scan of optic coherence
tomography (OCT) (left), color fundus image (center) and fluorescein angiography (FA) (right) of pretreatment (a), 3
months after SRT (b), resolution of SRF at 6 month (c), and final follow-up (at 12 month) (d). Subretinal fluid (SRF)
decreased and the leakage on FA disappeared at 3 months follow-up after SRT (b), followed by the SRF disappearance
at 6 months (c). At 12 months follow-up after SRT, there is no recurrence, and the outer retinal thickness wasincreased from 77 µm to 86 µm (d).

- 59 -

Hirayama et al

those both pre-treatment and at the time of resolution (pre-treatment vs final follow-up p＜0.01,
resolution vs final follow-up p＜0.01) (Fig. 3).
Mean CMT was 336±78 µm pre-treatment, 175±25 µm at resolution, and 185±28 µm at final
follow-up, with significant differences between all three time points (pretreatment vs resolution, p＜0.001; pretreatment vs final follow-up, p＜0.001; resolution vs final follow-up, p＜0.001) (Fig. 4).

There was no significant difference between IRT during at resolution (97±21 µm) and at final followup (98±24 µm; p＝0.45), whereas ORT showed a significant increase, where it was 77±11 µm at
resolution and 86±10 µm at final follow-up (p＜0.001) (Fig. 5).
Single correlation analysis identified pre-treatment BCVA, IRT at resolution, and the rates ofchange of IRT and ORT between at resolution and at final examination as significant factors

Figure 3. Box plots for the time course of best corrected visual acuity (BCVA) (logMAR). Mean BCVA was 0.01±0.22
pre-treatment, −0.04±0.20 at the time of resolution, and −0.10±0.15 at final follow-up, with significant improvement
between each time point toward the final follow-up.

Figure 4. Box plots for the time course of central macular thickness (CMT). Mean CMT was 336±78 µm pretreatment, 175±25 µm at resolution, and 185±28 µm at final follow-up, with significant differences between all threetime points.

- 60 -

Thickness of Retinal Layers after SRT

Figure 5. Box plots for the time course of inner retinal thickness (IRT) (a) and outer retinal thickness (ORT) (b).
There was no significant difference of IRT between at the time of resolution (97±21 µm) and at final follow-up (98±24µm), whereas ORT showed a significant difference, where it was 77±11 µm at the time of resolution and 86±10 µm at
final follow-up.

Table 2. Univariate analysis of factors associated with final BCVAr, ρp value

Sex＊1

-

0.50

Age

0.07

0.75

-

0.87

variables
Pretreatment＊2

Recurrence＊1
Duration of symptom

0.09

0.69

Pretreatment BCVA＊3

0.84＜0.001

Pretreatment CMT＊3−0.283

0.19＊2

Resolution
0.23

0.30

Inner retinal thickness＊3

−0.45

0.03

Outer retinal thickness

−0.39

0.07

Disappearance time of SRF＊2＊3

Final follow-up
Δ Inner retinal thickness＊3

−0.44

0.03

Δ Outer retinal thickness＊3−0.63＜0.001

1: Mann-Whitney U test. ＊ 2: Spearman's rank correlation coefficient. ＊ 3: Pearson's correlation coefficient.
BCVA, best corrected visual acuity; CMT, central macular thickness; and SRF, subretinal fluid.
＊

Table 3. Multivariate analysis of factors associated with final BCVA
variables

B

SE B

β

p value

BCVA

0.49

0.12

0.66

0.01

0.31

−0.37

0.03

Δ Outer retinal thickness
R2

−0.75

0.78

SE, standard error; and BCVA, best corrected visual acuity.

- 61 -

Hirayama et alassociated with final BCVA (Table 2). Stepwise regression analysis of factors for which p ＜0.20
identified significant associations with pre-treatment BCVA and the rate of change of ORT (Table 3).

Discussion
The persistence of SRF is known to cause structural changes in the neuroretina and RPE thatundoubtedly affect the prognosis for visual acuity. Atrophy of the subfoveal RPE and cystoid maculardegeneration have been implicated in causing the serious loss of vision that occurs in chronic CSC1,2).
However, more than that, the thickness of the outer nuclear layer (ONL) after resolution of SRF is
also considered to be one of the important determining factors for visual prognosis 18,19). When SRF
persists long-term, extension of the photoreceptor outer segment and thinning of the ONL are known
to occur 20,21). Regarding the retina of the patients with resolved CSC, Nakamura et al reported thatthe cone density (cell density in the ONL) measured with adaptive optics fundus camera issignificantly decreased, which is associated with the decrease of the ORT and visual acuity 22). In
cases of protracted SRF treated by photo dynamic therapy (PDT), the thickening of the ONL after
treatment is reportedly positively associated with the better visual prognosis after SRF
resolution18,19,23,24). These changes after SRF resolution following PDT include the improvement of thevisibility of the ellipsoid zone and the ONL increasing in thickness. In the present study, IRT, whichis almost the same as the range of the ONL at fovea, did not change significantly during postresolution follow-up, but the ORT increased significantly. In the detailed correlation analysis it wasalso shown that the thickness increase of the ORT after treatment is positively correlated to thevisual prognosis, not of the IRT, which is not consistent with the previous reports. As the reason of it,some possibility can be discussed; First, it might be just because the short observation period in thisstudy. Secondly, it may lie on the difference in the therapeutic mechanism between PDT and SRT.
Namely, it would be hypothesized that SRT might promote the restoration of photoreceptors outer
segment (the main component of the ORT), better or at least earlier, than after PDT. However, as
there are still no comparison studies, further investigation is necessary to evaluate the changes inretinal thickness after different therapies.
The limitations of this study include its nature as a retrospective study, its small sample size, thecomparatively short follow-up period of 3-6 months after SRF resolution, and the fact that patientswith recurrent or persistent SRF were excluded. If patients can be maintained long-term without
SRF recurrence, further changes in retinal structure might occur. The effect of recurrent or
persistent SRF must also be taken into account in the case of long-term follow-up.
In conclusion, SRT may improve final visual acuity significantly, and this improvement was
associated with two factors: pretreatment visual acuity and the rate of change in ORT. Although CSC
is a disease with a high tendency to heal spontaneously, the persistent SRF can cause themorphological changes in the neuroretina as well as of the RPE, which has been shown to associatewith the decreasing visual acuity. Therefore, its earlier resolution needs to be prioritized to preserve
the retinal structure and the long-term visual acuity of patients with CSC.
Acknowledgments
All authors have no COI to declare regarding the present study.

- 62 -

Thickness of Retinal Layers after SRT

References
1. Yannuzzi LA, Shakin JL, Fisher YL, et al. Peripheral retinal detachments and retinal pigment epithelial
atrophic tracts secondary to central serous pigment epitheliopathy. Ophthalmology 1984;91:1554-1572.
2. Spaide RF, Campeas L, Haas A, et al. Central serous chorioretinopathy in younger and older adults.
Ophthalmology 1996;103:2070-2080.
3. Loo RH, Scott IU, Flynn HW Jr, et al. Factors associated with reduced visual acuity during long-term followup of patients with idiopathic central serous chorioretinopathy. Retina 2002;22:19-24.
4. Piccolino FC, de la Longrais RR, Ravera G, et al. The foveal photoreceptor layer and visual acuity loss incentral serous chorioretinopathy. Am J Ophthalmol 2005;139:87-99.
5. Imamura Y, Fujiwara T, Spaide RF. Fundus autofluorescence and visual acuity in central serous
chorioretinopathy. Ophthalmology 2011;118:700-705.
6. Roider J, Liew SH, Klatt C, et al. Selective retina therapy (SRT) for clinically significant diabetic macularedema. Graefes Arch Clin Exp Ophthalmol 2010;248:1263-1272.
7. Brinkmann R, Roider J, Birngruber R. Selective retina therapy (SRT): a review on methods, techniques,preclinical and first clinical results. Bull Soc Belge Ophtalmol 2006;(302):51-69.
8. Roider J, Brinkmann R, Wirbelauer C, et al. Retinal sparing by selective retinal pigment epithelial
photocoagulation. Arch Ophthalmol 1999;117:1028-1034.
9. Schuele G, Elsner H, Framme C, et al. Optoacoustic real-time dosimetry for selective retina treatment. J
Biomed Opt 2005;10:064022.
10. Neumann J, Brinkmann R. Boiling nucleation on melanosomes and microbeads transiently heated bynanosecond and microsecond laser pulses. J Biomed Opt 2005;10:024001.
11. Park YG, Kim JR, Kang S, et al. Safety and efficacy of selective retina therapy (SRT) for the treatment ofdiabetic macular edema in Korean patients. Graefes Arch Clin Exp Ophthalmol 2016;254:1703-1713.
12. Roider J, Liew SH, Klatt C, et al. Selective retina therapy (SRT) for clinically significant diabetic macularedema. Graefes Arch Clin Exp Ophthalmol 2010;248:1263-1272.
13. Koinzer S, Elsner H, Klatt C, et al. Selective retina therapy (SRT) of chronic subfoveal fluid after surgery of
rhegmatogenous retinal detachment: three case reports. Graefes Arch Clin Exp Ophthalmol 2008;246:13731378.
14. Kang S, Park YG, Kim JR, et al. Selective retina therapy in patients with chronic central serous
chorioretinopathy: a pilot study. Medicine (Baltimore) 2016;95:e2524.
15. Elsner H, Pörksen E, Klatt C, et al. Selective retina therapy in patients with central serous chorioretinopathy.
Graefes Arch Clin Exp Ophthalmol 2006;244:1638-1645.
16. Framme C, Walter A, Berger L, et al. Selective retina therapy in acute and chronic-recurrent central serous
chorioretinopathy. Ophthalmologica 2015;234:177-188.
17. Yasui A, Yamamoto M, Hirayama K, et al. Retinal sensitivity after selective retina therapy (SRT) on patientswith central serous chorioretinopathy. Graefes Arch Clin Exp Ophthalmol 2017;255:243-254.
18. Matsumoto H, Sato T, Kishi S. Outer nuclear layer thickness at the fovea determines visual outcomes inresolved central serous chorioretinopathy. Am J Ophthalmol 2009;148:105-110.
19. Van Rijssen TJ, Mohabati D, Dijkman G, et al. Correlation between redefined optical coherence tomography
parameters and best-corrected visual acuity in non-resolving central serous chorioretinopathy treated withhalf-dose photodynamic therapy. PLoS One 2018;13:e0202549.
20. Matsumoto H, Kishi S, Otani T, et al. Elongation of photoreceptor outer segment in central serous chorioretinopathy.
Am J Ophthalmol 2008;145:162-168.
21. Ozdemir I, Eren A, Ersöz G. Outer nuclear layer thickness at the central fovea relation with symptom
duration in central serous chorioretinopathy. Int Ophthalmol 2018;18:doi:10.1007/s10792-018-0950-y.
22. Nakamura T, Ueda-Consolvo T, Oiwake T, et al. Correlation between outer retinal layer thickness and conedensity in patients with resolved central serous chorioretinopathy. Graefes Arch Clin Exp Ophthalmol 2016;
254:2347-2354.
23. Hua L, Lin B, Hong J, et al. Clinical research on one-third dose verteporfin photodynamic therapy in thetreatment of chronic central serous chorioretinopathy. Eur Rev Med Pharmacol Sci 2018;22:278-284.
24. Ohkuma Y, Hayashi T, Sakai T, et al. One-year results of reduced fluence photodynamic therapy for central
serous chorioretinopathy: the outer nuclear layer thickness is associated with visual prognosis. Graefes Arch
Clin Exp Ophthalmol 2013;251:1909-1917.

- 63 -

Osaka City Med. J. Vol. 65, 65-76, 2019

Assessment of Dual-time-point 18F-fluorodeoxyglucosePositron Emission Tomography/Computed Tomography Imaging of
Malignant Tumors Using Visually and Semiquantitatively:
What Kind of Tumors is Useful?
HIROTAKA SEURA1), TERUE OKAMURA1), KOICHI KOYAMA2), and YUTAKA MASUOKA3)
PET center1), Osaka Saiseikai Nakatsu Hospital;
Department of Diagnostic and Interventional Radiology2), Osaka City University
Graduate School of Medicine; and Department of Radiology3),
Osaka Prefectural Medical Center for Respiratory and Allergic Diseases

Abstract
BackgroundTo retrospectively evaluate the effectiveness of dual-time-point imaging 18F-fluorodeoxyglucosepositron emission tomography/computed tomography in assessing various malignant tumors at our
hospital.
Methods
This study included 1153 patients who underwent dual-time-point imaging from November 2009
to October 2011 (702 males, 451 females), including 1211 histopathologically diagnosed malignant
lesions. Early and delayed images were acquired at 1 and 2 h after intravenous injection,
respectively.

Fluorodeoxyglucose-positron emission tomography/computed tomography images were

evaluated visually and semiquantitatively.
Results
According to visual analysis, abnormal uptake was detected in 1032 and 1096 lesions among all
tumors, and 362 and 416 among 526 small tumors (diameter ＜3 cm) in early and delayed images,
respectively. On delayed images, abnormal uptake was detected in all lesions showing abnormal
uptake in early images, and 64 additional lesions among all tumors and 54 additional lesions amongsmall tumors that had not been detected in early images. Tumors detected in delayed images and notin early images included laryngeal, bile duct, pancreatic, uterine cervical and uterine corpus cancers.
In semiquantitative analysis, maximum standardized uptake values differed significantly between
early and delayed images in both all tumors and small tumors. Retention index, grouped as ＞10%,≧−10% but ≦10%, and ＜−10% were 942, 135 and 14 in all tumors, and 320, 86 and 13 in small

tumors, respectively.
Received May 25, 2018; accepted January 15, 2019.
Correspondence to: Hirotaka Seura, MD.
PET center, Osaka Saiseikai Nakatsu Hospital,
2-10-39, Shibata, Kita-ku, Osaka 530-0012, Japan
Tel: +81-6-6372-0712; Fax: +81-6-6372-0732
E-mail: hirotakaseura@yahoo.co.jp- 65 -

Seura et al

Conclusions
Dual-time-point imaging could be effective when fluorodeoxyglucose uptake is not detected inearly images, particularly in tumors with diameter ＜3 cm.
Key Words: Dual-time-point imaging; FDG-PET/CT; Malignancy; Standardized uptake
value (SUV)

Introduction
In recent years, F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed
18

tomography (CT) has been widely used as a common diagnostic tool for assessing tumor malignancy,
determining the clinical stage of tumors, judging therapeutic effects, and detecting recurrence. FDGPET/CT has been recognized as a useful diagnostic tool for various malignant tumors, but is known
to show some limitations. Differentiation between malignant tumors and benign lesions by FDG
accumulation alone is known to be difficult, because FDG accumulates in not only malignant tumors,
but also benign lesions including benign tumors and inflammatory lesions.
Dual-time-point imaging (DTPI) FDG-PET/CT has recently been recognized as an excellent andsimple tool for improving the diagnostic impact of FDG-PET/CT1-4). For this method, PET is usually
performed twice, at 1 h (as an early image) and approximately 2 h (as a delayed image) after
intravenous injection of FDG. Most malignant tumors show gradually increasing FDG uptake astime advances, whereas benign lesions usually show decreased or unchanged FDG uptake1,4,5). Using
this difference, DTPI offers higher diagnostic utility than standard FDG-PET/CT.
Previous reports, including meta-analyses, have assessed the usefulness of DTPI1,2,6). However, nodetailed assessments appear to have investigated DTPI for different malignant tumors using thesame PET system. Early and delayed images have usually been compared using standard uptake
values (SUV)1,3,6-8), but no investigations have compared visual analyses.
The purpose of this single-institution study was to retrospectively evaluate the effectiveness of
DTPI in visually and semiquantitatively assessing malignant tumors, and to investigate what kind oftumor DTPI is best suited to.

Methods
Patients
A total of 1153 consecutive patients on whom DTPI had been performed from November 2009 to
October 2011 were investigated (702 males, 451 females; age range, 17-90 years; median, 68 years).
Patients were diagnosed histopathologically, and in total were shown to have 1211 malignant lesions.
Numbers of malignant lesions found per location are shown in Table 1.
The software of PET reconstruction for our PET/CT scanners was updated in 2012. There were
the obvious differences between before and after software updated images in not only visual analysis
but also semi-quantitative analysis, therefore the cases after software updated were not included inthis study.
This study was approved by the ethics review board of our hospital (institutional review board
number: #H28-051) and was performed in accordance with the ethical standards of the 1964
Declaration of Helsinki and its later amendments.
FDG-PET/CT scanning
Images were acquired with a PET/CT single helical scanner (SET-3000BCT/L; Shimadzu, Kyoto,- 66 -

Dual-time-point FDG-PET/CT of Malignant Tumors
Table 1. The number of each malignant lesion

Japan) after the patient had fasted for a minimum of 5 h. Patients were administered FDG at 2.7
MBq/kg body weight and scanned at both 60 and 120 min after injection. Both early and delayedemission images covered the body of the patient from the orbits to the upper thighs and were obtainedby three-dimensional acquisition using a z-axis 20-cm field of view. Transmission scans were
simultaneously obtained using an external source (Cs-137). Table speed was 1 mm/s. Raw emission
images were reconstructed using the Fourier rebinning-dynamic row-action maximum likelihood
algorithm, and attenuation was corrected by the measured attenuation correction method. FDG was
produced by a cyclotron (CYPRIS-HM12; Sumitomo Heavy Industries, Tokyo, Japan). CT was
performed after completion of the delayed PET, using the same field of view to reconstruct PET/CT
images. CT parameters used were: peak energy, 120 kV; electric current, 60-180 mA according topatient body shape with appropriate radiation exposure; helical pitch, 7 mm; collimation, 7 mm;- 67 -

Seura et al

thickness, 7 mm. During CT, patients were requested to perform shallow breathing.
Image analysis
1. Image evaluation
All images were retrospectively reviewed by 2 radiologists, both with more than 10 years ofexperience. Consensus was reached by discussion.
2. Visual analysis
For all malignant tumor lesions visually detected in either early or delayed images, the observedchange in FDG uptake was assigned one of three possible labels: increasing; unchanged; ordecreasing. This study defined small malignant tumors as lesions ≦3 cm in diameter, because
Ohtaka et al reported that using maximum SUV (SUVmax) corrected by a recovery coefficient curve
calculated by measuring sphere activity and dividing that activity by true sphere activity was usefulin diagnosing lung cancer lesions ≦3 cm in diameter9). Referring to the recovery coefficient curve
described in the FDG-PET/CT imaging procedure guidelines for cancers (2nd edition) 10), SUV is
underestimated in lesions ≦3 cm because the recovery coefficient is ＜1.0. On the other hand, if thetumor is ＞3 cm, the recovery coefficient value approaches 1.0 and SUV is considered to be evaluatedaccurately. We examined the observed changes in FDG uptake not only for all malignant lesions, butalso for small malignant lesions alone.
3. Semiquantitative analysis
Semiquantitative analysis was performed using the SUVmax of malignant tumors. All malignant
tumors detected in either early or delayed images and with SUVmax that did not exceed themeasurable upper limit were analyzed.
The retention index (RI) compares FDG uptake in the early image to FDG uptake in the delayed
image and was calculated for each lesion. RI was calculated using the following formula: RI (%) ＝(SUVmax of delayed image−SUVmax of early image)/SUVmax of early image×100%.
Suitable RI thresholds have been previously reported as −10% and 10%11,12). Malignant tumors
were therefore divided into 3 groups using the calculated RI: RI ＜−10%; RI between −10% and 10%;and RI ＞10%. The same three categories were applied to the small malignant lesions group.
Statistical analysis
Wilcoxon signed-rank test was used to compare SUVmax between early and delayed images. For
all analyses, values of p＜0.05 were considered statistically significant.

Results
Visual analysis
The results of visual analyses for all 1211 malignant lesions, including details of the locations in
the early or delayed images, are shown in Table 2.
1. Lesion detection
A total of 1032 lesions (85.2%) and 1096 lesions (90.5%) were detected in early and delayed images,
respectively. All lesions detected on early images were also detected on delayed images. Of the 179
lesions (14.8%) not detected on early images, 64 lesions (35.8%) were detected on delayed images.
Typical cases with malignant tumors ＞3 cm in which delayed images were useful for detecting
tumors compared to the early image are shown in Figure 1.
Among all lesions, 526 lesions showed a diameter ＜3 cm. Of these, 362 lesions (68.8%) and 420
lesions (79.8 %) were detected on early and delayed images, respectively. All lesions detected on early- 68 -

Dual-time-point FDG-PET/CT of Malignant Tumors
Table 2. ‌The detectability of malignant lesions in the early image and the delayed image on the visual
analysis

images were also detected on delayed images. Of the 164 lesions (10.2%) not detected on early
images, 58 lesions (35.4%) were detected on delayed images.
Most lesions not detected on early images but detected on delayed images showed diameter ＜3
cm. Typical cases with malignant tumors ≦3 cm in which delayed images were useful for detecting
tumors compared to the early image are shown in Figure 2.
2. Lesion detection per location
Lesions detected on neither early nor delayed images were most frequently head and neck cancers(such as laryngeal, oral floor, thyroid, or tongue cancer), abdominal cancers (such as hepatic or
gallbladder cancer), breast cancers, and gastric cancers.
Lesions not detected on early images but detected on delayed images were frequently laryngeal
cancer, abdominal cancers (such as bile duct or pancreatic cancer), and gynecological cancers (such as
uterine cervical or uterine corpus cancer). On very few occasions, these lesions were from a cancer ofa digestive organ.
All lesions of gingival, nasal cavity and paranasal sinus cancer, epipharyngeal cancer, and morethan 90% of oropharyngeal, lung and colon cancer were detected on both early and delayed images.
Six lesions larger than 3 cm in diameter were not detected on early images, but all these lesions
were detected on delayed images. These lesions were tongue, hepatic, bile duct, ovarian, and lung- 69 -

Seura et al

Figure 1. Cancer lesions with diameter ≧3 cm.
a-d) A 67-year-old man with lung cancer (3 cm). The lesion could not be detected on the early image but was detectedon the delayed image. a，Early image from FDG-PET (SUVmax 1.2); b, Delayed image from FDG-PET (SUVmax1.8); c, Delayed image from FDG-PET/CT; and d, CT image.
e-h) A 62-year-old woman with uterine cervical cancer (3.7 cm). The lesion could be detected on both early and delayed
images, showing increased FDG uptake on the delayed image. e, Early image from FDG-PET, SUVmax 3.5; f,
Delayed image from FDG-PET, SUVmax 4.6; g, Delayed image from FDG-PET/CT; and h, MR image.

- 70 -

Dual-time-point FDG-PET/CT of Malignant Tumors

Figure 2. Cancer lesions with diameter ＜3 cm.
a-d) A 66-year-old man with bile duct cancer. This lesion could not be detected on the early image, but was detectableon the delayed image. a, Early image from FDG-PET (SUVmax 1.8); b, Delayed image from FDG-PET (SUVmax3.2); c, Delayed image from FDG-PET/CT; and d, CT image.
e-h) A 70-year-old man with laryngeal cancer (1.5 cm), this lesion was detected of both early and delayed images, andshowed increasing FDG uptake on delayed image; e, Early image (SUVmax 3.3); f, Delayed image (SUVmax 4.5); g,
Delayed image of FDG-PET/CT image; and h, CT image.

- 71 -

Seura et alcancers, and multiple myeloma.
3. Changes in FDG uptake between early and delayed images
Within head and neck cancers, gingival, epipharyngeal, and oropharyngeal cancers displayed thelowest percentage of lesions that showed increasing FDG uptake. Hypopharyngeal and laryngeal
cancers tended to show both increasing and decreasing changes. Other cancers (except hepatic
cancer) tended to show increasing FDG uptake. Increasing FDG uptake was observed more
frequently in lesions with diameter ＜3 cm than in all lesions (Table 3).
Semiquantitative analysis
1. SUVmax of all malignant lesions
A total of 1091 lesions were included in this part of the analysis. Because the remaining 120
lesions showed faint or no abnormal uptake, SUVmax of these lesions was too difficult to measure.
Mean (±standard deviation) SUVmax was 7.0±5.2 in early images and 8.8±6.4 in delayed images,
representing a significant difference (p＜0.0001).
Among these 1091 lesions, 420 lesions showed diameter ＜3 cm. For these lesions, SUVmax was
3.8±3.4 in early images and 4.7±4.1 in delayed images, again representing a significant difference (p＜0.0001).

2. RI of all malignant lesions

Table 3. ‌The changes between FDG uptake in the early images and that in delayed image on the visual
analysis

Increasing, higher FDG uptake on delayed image than early Image; no change, no significant change between early and delayed image; decreasing, lower
FDG uptake on delayed image than on early image; and N/A, not applicable.

- 72 -

Dual-time-point FDG-PET/CT of Malignant Tumors

A total of 942 lesions (86.3%) showed RI ＞10%, 135 lesions (12.4%) showed RI between −10% and10%, and 14 lesions (1.3%) showed RI ＜−10% (Table 4). Of the 420 lesions with diameter ＜3 cm, 321
lesions (76.4%) showed RI ＞10%, 86 lesions (20.5%) showed RI between −10% and 10%, and 13
lesions (3.1%) showed RI ＜−10% (Table 4).
3. RI per location
Most sites of all tumor lesions and small lesions showed RI ＞10%, including all nasal cavity andparanasal sinus cancers, hepatic cancers, multiple myelomas, and prostatic cancers. Among cancer
lesions with RI between −10% and 10%, the ear canal cancer was the most frequent site (4/6, 66.7%).
Fourteen lesions showed RI ＜−10% in all tumor lesions. These lesions comprised 2 hypopharyngeal
cancers, 2 laryngeal cancers, 2 breast cancers, 1 tongue cancer, 1 oropharyngeal cancer, 1 thyroid
cancer, 1 lung cancer, 1 esophageal cancer, 1 gastric cancer, 1 colon cancer and 1 malignant
lymphoma. Of the 14 lesions above, 13 lesions (excluding 1 case of colon cancer) showed diameter ＜3
cm (Table 4).
In cases where lesions showed diameter ＜3 cm, hypopharyngeal and breast cancers frequently
showed RI ＜−10% (Table 4).

Discussion
The utility of DTPI as a diagnostic tool has been reported previously, with most studies focusing

Table 4. Retention index of malignant lesions on the semiquantitative analysis- 73 -

Seura et alon the differentiation of malignant and benign tumors 1,4,6,11). No reports have investigated whichmalignant tumors benefit most from DTPI, as in the present study. Routine performance of DTPI in
all patients has recently been reported as inappropriate13), as dose exposure is increased for every CT
acquisition on the PET/CT scanner. Imaging of the target organ alone is thus becoming morecommon with DTPI. With the particular PET/CT scanner used in the present study, acquisition ofimages without unnecessarily increasing the exposure is possible while comparing early and delayed
images on an equal basis. We therefore visually and semiquantitatively investigated the kinds ofmalignant tumor for which DTPI is most useful using only patients scanned at our hospital.
In this study, the detection rate by visual analysis of all malignant tumor lesions was 85.2% on
early images and 90.5% on delayed images. This result revealed that delayed images were better
than early images for detecting malignant lesions. The detectability of malignant tumor lesions has
been reported as 77%-78% on early images and 94% on delayed images, comparable to our results1,2).
In small lesions ＜3 cm in diameter, detection rate was 68.8% on early images and 79.8% on delayed
images. These detection rates were lower than those corresponding to rates for the groupencompassing all lesions. Most lesions not detected on early images showed diameter ＜3 cm. Only
15 of 685 lesions (2%) with a diameter ≧3 cm were not detected on early images. Of the lesions withdiameter ＜3 cm not detected on early images, approximately one-third were detected on delayed
images. DTPI has been reported as useful for small pancreatic cancers, with an average diameter of
2.5 cm14). We suppose that DTPI would be useful for lesions with diameters ＜3 cm.
With regard to the utility of DTPI for different types of tumor, tumor lesions that were difficult to
detect on both early and delayed images were more frequently head and neck cancers, abdominal
malignant tumors and gastric cancers than other types of tumor lesions. Lesions that could not be
detected on early images but were detected on delayed images were only infrequently digestive tract
cancers, but were commonly laryngeal cancers, bile duct cancers, pancreatic cancers, uterine cervicalcancers, and cancers of the uterine corpus. Most of these tumors showed diameter ＜3 cm. The utilityof DTPI for head and neck cancers has been reported previously1,15). However, Yen et al reported that
detection rates of epipharyngeal cancer were higher with FDG-PET than with MRI, and no usefuladditional information was obtained from delayed imaging16). Various reports have examined DTPI in
thyroid cancer1,17,18), with limited success, and no reports have examined DTPI for laryngeal cancer. In
the present study, while DTPI seemed useful, some laryngeal cancer lesions failed to be detected by
FDG accumulation in both early and delayed images, possibly due to tumor disappearance orshrinkage after biopsy. DTPI has already proven useful in detecting bile duct cancer1,19), and reports
have been conflicting regarding differentiation of benign lesions from malignant pancreatic cancers
using delayed imaging20,21). SUVmax of the pancreatic adenocarcinoma was reported to increase
statistically from early to delayed time22). In the present study, detecting abnormal FDG uptake on
early images was difficult for these lesions due to the nature of the tumors and the small size. DTPI
proved useful in detecting both uterine cervical cancer lesions and lesions of the uterine corpus cancer
in the present study, but reports are conflicting on the usefulness of DTPI for uterine cervicalcancer23,24) and no reports have examined the situation for cancer of the uterine corpus. In the present
study, presence or absence of tumor FDG uptake was determined not only using SUVmax, but also by
visually analyzing tumor contrast compared to uptake by surrounding tissue FDG. To the best of ourknowledge, no other reports discussing the usefulness of DTPI have investigated tumor FDG uptake
in such a manner. Most tumor lesions showed visually increased uptake of FDG. This was attributed- 74 -

Dual-time-point FDG-PET/CT of Malignant Tumors

to increased tumor FDG uptake in delayed images and decreased uptake in the surrounding tissue
over time. In head and neck cancers, the proportion of lesions showing increasing FDG uptake wassmall in cases of gingival cancer, epipharyngeal cancer and oropharyngeal cancer among small tumor
lesions. In these tumors, the utility of DTPI has been considered limited. Based on the above results,
we strongly recommend DTPI as a useful tool for detecting laryngeal cancer, bile duct cancer,
pancreatic cancer, uterine cervical cancer, and cancer of the uterine corpus, particularly for small
tumors.
In semiquantitative analysis, SUVmax was significantly increased on delayed images compared toearly images for both all lesions and lesions smaller than 3 cm. This finding is consistent with resultsfrom previous reports1,2,4).
Concerning RI, 86.3% of all lesions showed RI ＞10%. For tumors with diameter ＜3 cm, the threegroups of RI ＞10%, −10%＜ RI ＜10% and RI ＜−10% were almost equivalent to the three groups ofincreasing, unchanged and decreasing uptake on visual analysis. For all tumors, the group with RI＜−10% (1.3%) was almost equivalent to the group with decreasing FDG uptake on visual analysis(1.2%), but the group with RI ＞10% (86.9%) was larger than the group with increasing FDG uptake
on visual analysis (68.9%). This suggests that visually assessing the increase in delayed images isdifficult because of the high uptake in large tumors seen even on early images.
Of the only 1.3% of total tumor lesions showing a reduction of ＞10% in SUVmax, most showed adiameter ＜3 cm. The hypopharyngeal and breast cancers were included among these lesions. No
reports have discussed RI for hypopharyngeal cancer. Our results for the RI of breast cancer weresimilar to those from other published reports1).
Within the head and neck cancer group, the proportion of tumor lesions showing increased RI washigh among laryngeal, nasal and paranasal cavity cancers. All lesions of the bile duct, pancreatic,
uterine cervix and uterine corpus showed increased RI.
The findings of the present study must be considered in light of various limitations. First, thisstudy only analyzed malignant lesions, and benign tumors and inflammatory lesions were notincluded. Second, since this study was retrospective in design, various biases may have been present
despite the inclusion of consecutive patients.
In conclusion, additional delayed imaging appears warranted if tumor FDG uptake cannot be
detected on early images, particularly for tumor lesions with diameter ＜3 cm. Regarding head andneck cancers, DTPI is not thought to be necessary for hypopharyngeal cancer or oral floor cancer, but
additional delayed imaging is recommended to be performed for laryngeal cancer. For tumors otherthan head and neck tumors, additional delayed imaging is recommended to be performed for cancersof the bile duct, pancreas, uterine cervix, and uterine corpus.
Acknowledgments
All authors have no COI to declare regarding the present study.
References
1. Schillaci O. Use of dual-point fluorodeoxyglucose imaging to enhance sensitivity and specificity. Semin Nucl
Med 2012;42:267-280.
2. Chan WL, Ramsay SC, Szeto ER, et al. Dual-time-point 18F-FDG-PET/CT imaging in the assessment ofsuspected malignancy. J Med Imaging Radiat Oncol 2011;55:379-390.
3. Cui J, Zhao P, Ren Z, et al. Evaluation of dual time point imaging 18F-FDG PET/CT in differentiating- 75 -

Seura et al
malignancy from benign gastric disease. Medicine (Baltimore) 2015;94:e1356.
4. Cheng G, Torigian DA, Zhuang H, et al. When should we recommend use of dual time-point and delayed timepoint imaging techniques in FDG PET? Eur J Nucl Med Mol Imaging 2013;40:779-787.
5. Cheng G, Alavi A, Lim E, et al. Dynamic changes of FDG uptake and clearance in normal tissues. Mol Imaging
Biol 2013;15:345-352.
6. Saleh Farghaly HR, Mohamed Sayed MH, Nasr HA, et al. Dual time point fluorodeoxyglucose positronemission tomography/computed tomography in differentiation between malignant and benign lesions in cancer
patients. Does it always work? Indian J Nucl Med 2015;30:314-319.
7. García Vicente AM, Soriano Castrejón Á, León Martín A, et al. Molecular subtypes of breast cancer: metabolic
correlation with 18F-FDG PET/CT. Eur J Nucl Med Mol Imaging 2013;40:1304-1311.
8. Lin WY, Tsai SC, Hung GU. Value of delayed 18F-FDG-PET imaging in the detection of hepatocellular
carcinoma. Nucl Med Commun 2005;26:315-321.
9. Ohtaka K, Hida Y, Kaga K, et al. Outcome analysis of (18)F-fluorodeoxyglucose positron-emission tomography
in patients with lung cancer after partial volume correction. Anticancer Res 2013;33:5193-5198.
10. Hiroyoshi H, Keiichi O, Takahiro S, et al. Gan FDG-PET/CT satsueihou gaidolain dai2han, Kakuigaku
Gijutsu 2013;33:377-420. (In Japanese)
11. Lan XL, Zhang YX, Wu ZJ, et al. The value of dual time point (18)F-FDG PET imaging for the differentiation
between malignant and benign lesions. Clin Radiol 2008;63:756-764.
12. Sanz-Viedma S, Torigian DA, Parsons M, et al. Potential clinical utility of dual time point FDG-PET for
distinguishing benign from malignant lesions: implications for oncological imaging. Rev Esp Med Nucl 2009;28:159-166.
13. Lee ST, Scott AM. Are we ready for dual-time point FDG PET imaging? J Med Imaging Radiat Oncol 2011;
55:351-352.
14. Kawada N, Uehara H, Hosoki T, et al. Usefulness of dual-phase 18F-FDG PET/CT for diagnosing small
pancreatic tumors. Pancreas 2015;44:655-659.
15. Hustinx R, Smith RJ, Benard F, et al. Dual time point fluorine-18 fluorodeoxyglucose positron emission
tomography: a potential method to differentiate malignancy from inflammation and normal tissue in the headand neck. Eur J Nucl Med 1999;26:1345-1348.
16. Yen TC, Chang YC, Chan SC, et al. Are dual-phase 18F-FDG PET scans necessary in nasopharyngeal
carcinoma to assess the primary tumour and loco-regional nodes? Eur J Nucl Med Mol Imaging 2005;32:541548.
17. Lee S, Park T, Park S, et al. The clinical role of dual-time-point 18F-FDG PET/CT in differential diagnosis of
the thyroid incidentaloma. Nucl Med Mol Imaging 2014;48:121-129.
18. D'Souza MM, Marwaha RK, Sharma R, et al. Prospective evaluation of solitary thyroid nodule on 18F-FDG
PET/CT and high-resolution ultrasonography. Ann Nucl Med 2010;24:345-355.
19. Nishiyama Y, Yamamoto Y, Kimura N, et al. Comparison of early and delayed FDG PET for evaluation ofbiliary stricture. Nucl Med Commun 2007;28:914-919.
20. Nakamoto Y, Higashi T, Sakahara H, et al. Delayed (18)F-fluoro-2-deoxy-D-glucose positron emission
tomography scan for differentiation between malignant and benign lesions in the pancreas. Cancer 2000;
89:2547-2554.
21. Santhosh S, Mittal BR, Bhasin D, et al. Dual-phase 18F-FDG PET/CT imaging in the characterization of
pancreatic lesions: does it offer prognostic information? Nucl Med Commun 2014;35:1018-1025.
22. Mena E, Sheikhbahaei S, Taghipour M, et al. 18F-FDG PET/CT metabolic tumor volume and intratumoral
heterogeneity in pancreatic adenocarcinomas: impact of dual-time point and segmentation methods. Clin Nucl
Med 2017;42:e16-e21.
23. Yen TC, Ng KK, Ma SY, et al. Value of dual-phase 2-fluoro-2-deoxy-d-glucose positron emission tomography incervical cancer. J Clin Oncol 2003;21:3651-3658.
24. Yu L, Jia C, Wang X, et al. Evaluation of 18F-FDG PET/CT in early-stage cervical carcinoma. Am J Med Sci
2011;341:96-100.

- 76 -

Instructions for Authors
The Osaka City Medical Journal will consider the publication of any original manuscript,
review, case report, or short communication. Articles should be in English.
Manuscript submission. Manuscripts should be sent to the Editor, Osaka City Medical Journal,
Osaka City Medical Association, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka
545-8585, Japan; phone and fax 06-6645-3782; e-mail shiigakukai@med.osaka-cu.ac.jp
The Journal accepts only manuscripts that contain material that has not been and will not be publishedelsewhere. Duplicate publication of scientific data is not permitted. If closely related papers might beconsidered to be duplicate publications, the possible duplicate should be submitted with the manuscript andthe authors should explain in their covering letter what is original in the submitted paper. Submit threecopies of the manuscript (two of which may be photocopies) together with CD-R or DVD-R containing the
body text, tables and figures. The manuscript should be prepared by Microsoft Word or its compatible
software (doc or docx). Acceptable formats for figures are JPG or TIF. Use only 12-point font size and astandard serif font. Double-space throughout the manuscript and use standard-sized (ISO A4, 212×297mm) white bond paper. Make margins at least 25 mm wide on all sides. Number pages consecutively
starting with the title page and ending with the reference list. Begin each of these sections on a separate
page: title page, abstract, text, acknowledgments, references, tables (each one separate), and figure legends.
Do not use abbreviations in the title or abstract and limit their use in the text, defining each when it first
appears. Manuscripts should meet the requirements outlined below to avoid delay in review andpublication. Authors whose native language is not English must seek the assistance of a native English
speaker who is familiar with medical sciences. Please attach the certificate from the person(s) who edit themanuscript. Some minor editorial revision of the manuscript will be made when the editorial committee
considers it necessary.

Title page. All submissions must include a title page. The full title of the paper, should be concise,

specific, and informative, and should contain the message of the paper without being in sentence form.
Next, include the full names and academic affiliations of all authors, and indicate the corresponding author,
address, phone, fax, and e-mail address. Give a running title (not to exceed 50 characters including spaces),and three to five key words. Last, give the word count for text only, exclusive of title, abstract, references,tables, and figure legends.

Structured abstract. The abstract of 250 words or less should consist of four paragraphs headed
Background, Methods, Results, and Conclusions.
Text. Full papers about experiments or observations may be divided into sections headed Introduction,
Methods, Results, and Discussion.
Tables. Each table should be typed on a separate sheet in characters of ordinary size, double-spaced(with at least 6 mm of white space between lines). Each table must have a title and should be assigned an
Arabic numeral ( Table 3 ). Vertical rules should not be used.

Figures. For black-and-white figures, submit three original glossy prints or laser-quality proofs andthree photocopies of each. One transparency and three color prints should be submitted of each color figure.
Label the front of figures with the figure number. Indicate on the back of each figure the first author, thefirst few words of the manuscript title, and the direction of the top of the figure (if needed).
Photomicrographs should have scale markers that indicate the magnification. Provide figure legends on a
separate page, double-spaced, immediately after the tables. All illustrations and graphs, to be referred to as
figures, should be numbered in Arabic numerals ( Fig. 2 etc.). The approximate position of each figure in
the text should be indicated in the right margin of the manuscript. Illustrations in full color are acceptedfor publication if the editors judge that color is necessary, with the cost paid by the author.

References. Reference must be double-spaced and numbered consecutively in the order cited in the

text. When listing references, follow the style of the Uniform Requirements (http://www.icmje.org/) andabbreviate names of journals according to PubMed (http://www.ncbi.nlm.nih.gov/sites/netrez). List all
authors up to three; when there are four or more, list the first three and use et al.
Examples of reference style
1. Priori SG, Schwartz PJ, Napolitano C, et al. Risk stratification in the long-QT syndrome. N Engl J Med
2003;348:1866-1874.
2. Schwartz PJ, Priori SG, Napolitano C. The long-QT syndrome. In: Zipes DP, Jalife J, editors. Cardiac
electrophysiology: from cell to bedside. 3rd ed. Philadelphia: W.B. Saunders; 2000. pp. 597-615.

Proofs. One set of proofs together with the original manuscript will be sent to the author, to be carefully

checked for any essential changes or printer's errors. The author is requested to return the corrected proofs
within 48 h of their receipt.

Short communications and case reports.

1. A short communication should have between 1500 and 2000 words, including the abstract. This word
count is equivalent to about four double-spaced manuscript pages.
2. The original and two copies including three sets of figures and tables should be sent to the Editorial
Office.

Manuscript submission fee. A nonrefundable fee of 10,000 JPY is due on submission of original
manuscripts, case reports, and short communications. A manuscript returned beyond six months of thedate of the initial decision will be considered a new submission.
Page charges and reprints. Authors are required to pay page charges of 10,000 JPY per printed
page to share in the high costs of publication. Authors receive with the proofs a reprint order form thatmust be filled out and returned with the proofs to the Editorial Office. Later orders cannot be filled because
reprints are made at the time the journal is printed.
Studies of human subjects. It is the responsibility of the authors to ensure that any clinicalinvestigation they did and report in manuscripts submitted to the Osaka City Medical Journal are inaccordance with the Declaration of Helsinki (http://www.wma.net).
Animal studies. It is the responsibility of the authors to ensure that their experimental procedures
are in compliance with the Guiding Principles in the Care and Use of Animals (http://www.the-aps.org/
pub_affairs/humane/pa_aps_guiding.htm) published each month in the information for authors of theAmerican Journal of Physiology.
Conflict of Interest (COI) Disclosure.

1. It is the responsibility of the authors to disclose any COI by signing the form.
2. If the authors have no conflicts, please state All authors have no COI to declare regarding the present
study in the Acknowledgments.
Authors are required to disclose any relationships with company or organization (such as funds,
consultancy fee, grant, fee for speaking, stock or shares). At the time of initial submission, thecorresponding author is responsible for obtaining conflict of interest information from all authors.

[Reviced: April 2019]

COPYRIGHT TRANSFER AND STATEMENT OF ORIGINALITY
We approve the submission of this paper to the Osaka City Medical Association for publication and havetaken to ensure the integrity of this work. We confirm that the manuscript is original and does not in wholeor part infringe any copyright, violate any right of privacy or other personal or priority right whatever, or
falsely designate the source of authorship, and that it has not been published in whole or in part and is not
being submitted or considered for publication in whole or in part elsewhere (abstracts excluded).
We agree to transfer copyright the manuscript entitledto the Osaka City Medical Association upon its acceptance for publication.

Write clearly and signature(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)(Author: print or type)(Signature)(Date)OCMJ for Conflict of Interest (COI) Disclosure Statement
The purpose of this form is to provide readers of your manuscript with information about your otherinterests that could influence how they receive and understand your work. The form is designed to becompleted and stored electronically. Each author should submit this form and is responsible for theaccuracy and completeness of the submitted information.

1. 1) Have you accepted from a sponsor or any company or organization (More than 1,000,000 JPY per year
from a specific organization) ? (Please circle below)(1) Funds ? ····································································································································Yes / No
(2) Consultancy fee ? ····················································································································Yes / No
(3) Any grant ? ······························································································································Yes / No
(4) Fee for speaking ? ···················································································································Yes / No
2) Do you hold any stock or shares related to the manuscript ?
(1) Directly ? ·································································································································Yes / No
(2) Indirectly, via family members or relatives ? ········································································Yes / No3) If any of above items are “yes”, please provide detailed information below.

2. If none of the above apply and there is no COI please clearly state “All authors have no COI to declareregarding the present study”.

Manuscript Title:
Name / Signature
Manuscript Identifying Number (if you know it):

Date

Editorial Committee

Masaaki Inaba, MD (Chief Editor)
Yasuhiro Fujiwara, MD

Wakaba Fukushima, MD

Kazuo Ikeda, MD

Motoharu Imanaka, MD

Koki Inoue, MD

Norifumi Kawada, MD

Shoji Kubo, MD

Yukio Miki, MD

Yukio Nishiguchi, MD

Masahiko Ohsawa, MD

Toshiyuki Sumi, MD

Masahiro Tanaka, MD

Daisuke Tsuruta, MD

The volumes and issues published in 1954-2018 were as follows: Vol 1 (one issue), Vols 2-5 (2issues, each), Vols 6-7 (one issue, each), Vols 8-9 (2 issues, each), Vol 10 (one issue), Vol 11 (2issues), Vol 12 (one issue), Vol 13 (2 issues), Vols 14-20 (one issue, each), Vol 21 (2 issues), Vol 22(one issue), Vols 23-64 (2 issues, each)

Publisher : Osaka City Medical Association,
Osaka City University Medical School,
1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan