Jpn J Clin OncoI1998;28(lO)590-596
I
Original Articles I
Genotype-Phenotype Correlation of Patients with Multiple
Endocrine Neoplasia Type 2 in Japan
Shin-ichi Egawa1, Hitoyasu Futaml', Kayo Takasakll, Masatoshi Iihara2, Takahiro Okamoto2, Masako Kanbe2,
Toshitaka Ohi 3, Yukie Saio4, Akira Miyauchi5, Yumi Takiyama6, Masafumi Koga7, Ken Miyanaga8,
Ken-ichi Inoue9, Shoshu Mitsuyama10, Yoshio Nomura", Hiroyuki TakeP2, Soichi Mugiya13, Osamu Ishida14,
FUjio Zeze15, Shiro shakutsul", Hiroyuki lnoue", Hiroyuki Oya18, Akira Yoshimura 19, Shinji Ishizuka20,
Takeshi Tsujino21, Takao Obara2and Ken Yamaguchi 1
For author affiliations, please see p.596
Background: Multiple endocrine neoplasia type 2 (MEN 2) is a hereditary syndrome
characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and hyperparathyroidism. MEN 2 is caused predominantly by germ-line mutations of the RET proto-oncogene. This
study aimed to clarifythe genotype-phenotype correlation in MEN2 patients in Japan in orderto
modify the clinical management according to the genotype.
Methods: Constitutive DNAof 64 MEN 2 patients (48kindreds) were searched for mutations at exons
10, 11, 13, 14 and 16 of the RET proto-oncogene using polymerase chain reaction-single strand
conformation polymorphism (PCR-SSCP), direct sequencing and restriction enzyme digestion. The
clinical characteristics of the patients were obtained from a previous nationwide questionnaire survey.
Results: Overall, 62 (96.9%) out of 64 patients had a germ-line point mutation at the hot spots.
MTC and pheochromocytoma occurred equally in every genotype except C630S. Specific
genotypehada correlation between tumorsizeandageattheoperation for MTCor extentof MTC,
i.e. C618S developed lateonsettypeof MTCas compared withthat of C634R, C634Yand M918T.
Small MTC in C634R may be less aggressive than those in C634Y and M918T.
Conclusions: DNA testing has good clinical implications for the management of patients with
MEN 2 and the timing and operative procedures of thyroidectomy can be modified according to
the genotype.
Key words: multiple endocrine neoplasia - RET proto-oncogene - DNA testing - genotype-phenotype
correlation
INTRODUCTION
Multiple endocrine neoplasia type 2 (MEN 2) is a familial
disorder inherited in an autosomal dominant trait. Patients with
MEN 2A develop medullary thyroid carcinoma (MTC), pheochromocytoma and hyperplasia or adenoma of the parathyroid
gland with hyperparathyroidism (1). Patients with MEN 2B
develop MTC and pheochromocytoma but rarely parathyroid
Received April 16, 1998; accepted June 30, 1998
For reprints and all correspondence: Hitoyasu Futami, Growth Factor
Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome,
Chuo-ku, Tokyo 104, Japan
Abbreviations: MEN, multiple endocrine neoplasia; MTC, medullary
thyroid carcinoma; FMTC, familial MTC
disease and are associated with Marfanoid habitus, intestinal
ganglioneuromatosis and neurofibromatosis. Familial MTC
(FMTC) syndrome is defined as patients whose families have at
least four members with MTC alone, without a family history of
either pheochromocytoma or parathyroid disease. Any patients
with either MTC alone where screening for other organ
involvement was not performed or FMTC families with fewer
than four cases of MTC were classified as 'other' to minimize
confusion of disease phenotypes due to small family size or
incomplete screening information (2). In addition to familial
diseases, there are sporadic cases of MTC, pheochromocytoma
and hyperparathyroidism.
Recently, germ-line point mutations affecting different domains of the RET proto-oncogene on chromosome 1Oq 11.2 were
found to be associated with MEN 2A (3,4), 2B (5,6), FMTC (4)
and 'other' (2). Accordingly, several investigators have shown
JpnJ Clin OncoI1998;28(lO)
that prophylactic total thyroidectomy at an early age is a useful
intervention for management of MTC in :MEN 2 gene carriers
who are identified by RET mutational analysis (7,8). It is still
controversial, however, when and on whomprophylactic thyroidectomy should be performed. There has been no report which
compares the detailed clinical aggressiveness of MTC among
genotypes. At first,the clinical characteristics, naturalhistoryand
clinical outcome of patients in Japan were investigated by a
nationwide questionnaire survey (9). This survey revealed that
the prognosis of the disease depends predominantly upon the
extent of MTC and that an early stage diagnosis and surgeryare
important. Since the risk of complications of thyroidectomy in
young children should be counterbalanced by the gain from the
prevention of MTC, it should be clarified which genotypes
developan aggressive MTC at an early age. This is the first study
analyzingthe correlation between genotypeand clinicalaggressivenessofMTC in orderto adopt prophylacticoperationfor:MEN
2 patients in Japan.
591
exon 13,70°C for exon 14and 56°C for exon 16for 1.5min and
extension at 72°C for 1.5 min with an additional 5 min at 72°C.
The PCR product was then subjected to single strand conformation polymorphism (SSCP) analysis (12). Electrophoresis was
performed in at least two temperature conditions using an
electrophoretic apparatus with a circulating water-bath (Genoquencer SSCP, ATTO, Tokyo). Gels were dried and exposed to
X-ray film. PCR products showing single strand conformational
variants (SSCV) were subjected to direct sequencing using a
Sequenase II PCR product direct sequencing kit (United States
Biochemical, Cleveland, OH) according to the manufacturer's
instructions. If no mutation was detected by SSCP within these
five exons, the PCR productswere subjectedto directsequencing
to avoidfalse-negative resultsof SSCPanalysis. In thesepatients,
SSCP analysis of the remaining 16 exons was performed using
primers and conditions described previously (11). When the
mutationscreatedor destroyedrestriction sites,the PCRproducts
were digested by appropriate restrictionenzymes to confirm the
mutation. The digests were divided on 8% polyacrylamide gel
and autoradiographed.
MATERIALS AND METHODS
PATIENTS
GENOTYPE-PHENOTYPE CORRELATION
From January 1995 to April 1997, physicians working in the
fields of endocrinology, .surgery and urology were asked to
provide 5-10 ml of whole blood from patients with :MEN 2 with
informedconsentaboutthe benefits andrisksof DNAtesting. For
each patient, the following data were obtained: family history,
clinical features, biochemical data, location, size and other
clinicalcharacteristics ofMTC, pheochromocytoma andparathyroid lesions, operative procedures and treatment outcomes (9).
The patients with :MEN 2 included in this study were subclassified into four categoriesby clinical features::MEN 2A,:MEN 2B,
FMTC and 'other' as described above.
The genotype was defined and described as the amino acid
substitution created by the point mutation (13); for example,
either AGC or TCC mutation at codon 618 which substitutes
serine for cysteine (TGC) was describedas the same genotypeas
C6l8S. The penetration of MTC, pheochromocytoma and
hyperparathyroidism was defined as the fraction of the presence
of the disease at the time of the questionnaire. All the statistical
analyses were carried 04t with the Fisher (two-tailed) exact test
and Student's t-test using StatView 4.02 software (Abacus
Concepts, Berkeley, CA). Only patients with objective data
(pathologically confirmed tumor diagnoses) were included in
statistical analysis. In order to investigate the relationship
betweengenotype and the aggressiveness of the MTC, the tumor
size was plotted againstthe age at the operationfor MTC in each
genotype which had more than three informative cases. The
correlation between the size and the age at operation was
calculated using StatView software. The clinical aggressiveness
of the MTC was evaluated as the presence of lymph node
metastasis or persistent tumor after radical operation confirmed
by a positivecalcitonintest.Thephenotypeof thepheochromocytoma was represented by laterality, pathological findings and
elevated serum catecholamines.
DNA TESTING
Constitutive DNA was extracted from the blood of the patients
using a Qlamp blood kit (QIAGEN, Hilden, Germany), according to the manufacturer'sinstructions.The mutational 'hot spots'
expanding exons 10, 11, 13, 14 and 16 were amplified with
32P-Iabeled primers (10,11) using an automated thermal cycler
(Robocycler, Stratagene, La Jolla, CA). The polymerase chain
reaction (PCR) was performed by 30 cycles of denaturation at
96°C for 1 min, annealingat 68°C for exons 10and 11,66°C for
Table 1. Clinical subtypes and mutations in the RET proto-oncogene: number of kindreds
MEN2A
Exon 10
Exon 10
Exon II
Exon 16
codon 618
codon 630
codon 634
codon 918
6
33
2
MEN2B
No mutation
Total
2
41
3
FMTC
'Other'
2
3
592
Genotype andphenotype of MEN 2
Table 2. Penetration of MTC, pheochromocytoma, parathyroid disease and metastatic MTC in each genotype
genotype clinical subtype MTC penetrance pheochromocytoma parathyroid disease
C618S
I FMTC
5 MEN 2A
C618F
C618R
metastatic MTC
I MEN2A
2 MEN2A
C630S
2 'other'
C634R
20MEN2A
6/6 (49.0·· h)
2/6 (56.0)
0/6
1/4
•
0
N.I.
1/1 (51.0)
•
1/1 (47.0)
•
0
2/2 (4 I.5 a )
2/2 (36.5)
0/2
•
0
0
0
•
•
0/1
2/2
I'other'
21/21 (26.7)
C634G
I MEN2A
I 'other'
C634Y
18121 (31.7)
4121(22.0)
()
0
15/17(41.9)
2118(44.5)
•
C634S
2 MEN2A
C634F
5 MEN 2A
I MEN 2B
o
3 MEN 2B
mutation
2 MEN2A
()
•
4/5 (43.3)
0/5
3/4
•
•
0
N.I.
1/1 (N.I.)
011
(J
0
~
1/3 (16.0)
0/3
2/3
()
•
e
3/3 (11.7)
no
•
5/5 (42.Z·· h )
1/1 (15.0)
M918T
•
18 MENZA
16/17(38.2a,b)
C634W
11/18
()
1/2 (N.I.)
•
2/2 (62.0)
112(70.0)
III
Positive casesiinformative cases, the size of circle indicates the informative cases, black area of the circle
indicates the positive cases: N.l., no informative cases. (Mean age at the operation for MTC, pheochromocytoma and HPT, respectively.) ap < 0.01 vs M918T. bp < 0.05 vs C634R.
RESULTS
Out of 48 kindreds with MEN 2, 41 were subclassified as MEN
2A clinically, three as MEN 2B, one as FMTC and three as
'other'. Overall 46 (95.8%) out of 48 MEN 2 kindreds in Japan
had a germ-line mutation in the 'hot spots' of the RET
proto-oncogene (exons 10, 11, 13, 14 and 16, Table 1). Out of 41
MEN 2A kindreds, 33 had a point mutation at codon 634 in exon
11 and six at codon 618 in exon 10. All the mutations occurred at
the cysteine residues within the extracellular cysteine-rich
domain (14). No point mutation was detected by either SSCP or
direct sequencing within the hot spot in the two patients with
MEN 2A and no SSCV was detected in the remaining 16 exons
by SSCP analysis. Out of three MEN 2B kindreds, two possessed
a classical point mutation at codon 918 in exon 16, tyrosine kinase
domain 2 (14), which substitutes threonine for methionine, while
one kindred (one patient) whose clinical manifestation was
compatible with MEN 2B possessed a point mutation at codon
634 which substitutes tryptophan for cysteine (manuscript in
preparation). One FMTC kindred had a point mutation at codon
618 which substitutes serine for cysteine. One 'other' kindred
possessed a mutation at codon 630 which was found only in this
subtype and two at codon 634.
GENOTYPE-PHENOTYPE CORRELATION
In order to assess the relationship between RET mutations and
disease features, the association of mutation and the presence or
Jpn J Clin OncoI1998;28(10)
absence of MTC, pheochromocytoma and hyperparathyroidism
were examined (Table 2). Although there was no statistical
difference in the frequency of MTC, pheochromocytoma and
hyperparathyroidism among the genotypes, the age at the operation
for MTC was significantly different among the genotypes. Patients
with M918T and C634R were operated at a mean age of 11.7 years
old (y.o.) and 26.7 y.o. respectively, which is significantly younger
than the age at operation for C618S, C618R, C634Y and C634F
patients. The mean ages at the operation for pheochromocytoma
were not statistically different among the genotypes. There was
also not a genotype in which the mean age at the operation for MTC
was statistically different from that for pheochromocytoma. Two
females who had C634Y and C634S genotypes, respectively,
developed only pheochromocytoma. One female with no RET
mutation developed pheochromocytoma and hyperparathyroidism
but not MTC. Hyperparathyroidism was present mainly in the
patients with point mutation at codon 634, but the frequency was
not statistically different owing to the small number of cases. The
genotype C630S was found in one 'other' kindred (two patients)
who developed only MTC.
In order to distinguish the tumor aggressiveness associated with
the genotype, the size and extent of the MTC were plotted against
the age at operation. The correlation coefficient was calculated if
the genotype had more than three informative cases. C618S
genotype had a good correlation between size and age (R2 =
0.982). One C618S case had lymph node metastasis and residual
tumor after radical operation, but three other cases were cured by
the operation (Fig. lA). C634R genotype is the most frequent
type and the mean age at operation was 26.7 y.o. (the youngest
case was 8 y.o.). In this genotype, there was no correlation (R2 =
0.02) between the size and the age at the operation (Fig. IB), but
the tumor seems to stay within the thyroid' gland while the tumor
is small; i.e. six out of seven cases smaller than 2 em were cured
by radical operation, while eight out of nine cases larger than 2 cm
had metastatic disease (p < 0.01, Table 3). In contrast, MTCs of
the patients with C634Y were progressive (Fig. 1C). The mean
age at operation in C634Y cases was 39.9 y.o. (the youngest case
was 19 y.o.) with no correlation with the size (R2 = 0.064). Ten
out of 12 cases (83.3%) had metastatic disease and four out of five
patients had metastatic disease even when the primary tumor was
smaller than 2 em in diameter (Table 3). The classical MEN 2B
genotype, M918T, developed large and aggressive MTC at an
early age with a strong correlation (R2 = 0.951, Fig. ID). The
mean age at operation for MTC in MEN 2B patients was 11.7 y.o.
(the youngest case was 8 y.o.). An 11 y.o. girl and a 16 y.o. boy
received radical operation for 2.0 and 4.0 ern MTCs, respectively,
but the following provocative calcitonin test revealed that they
had residual disease.
The phenotype of pheochromocytoma varied according to the
genotype (Table 4). No pheochromocytoma developed in the
C630S genotype. The laterality of involved adrenal gland was not
different among the genotypes. Eleven out of 12 C634R and eight
out of 12 C634Y genotypes developed multicentric lesions. No
malignant pheochromocytoma was found. Ectopic pheochromocytoma developed only in a 53 y.o. male patient in whom no
RET mutation was detected. The serum noradrenalin level was
elevated only in two patients with no mutation, while adrenalin
or both adrenalin and noradrenalin levels were elevated in the
patients with C618 or C634 mutations. One patient with M918T
genotype developed pheochromocytoma with a normal serum
catecholamine level.
DISCUSSION
The detailed phenotypes of 64 patients (48 kindreds) with MEN 2
in Japan were compared for the first time according to the
mutational analysis of the RET proto-oncogene. Overall, more than
95% of the patients possessed a mutation within the reported 'hot
spots' of the RET proto-oncogene encoding extracellular cysteinerich domain and tyrosine kinase domain. No point mutation was
detected within the entire coding region of the RET proto-oncogene
in two MEN 2A patients whose clinical manifestations were
compatible with the criteria of MEN 2A, i.e. one male developed
both MTC and ectopic pheochromocytoma and one female
developed pheochromocytoma and hyperparathyroidism with her
brother developing MTC. Neither of them had ganglioneuromatosis. The International RET Mutation Consortium (2) collected 477
MEN 2 kindreds and reported four out of 203 MEN 2A kindreds
without RET mutation. These data, including this study, suggest
some causes of the negative results of the mutational search. One
may come from the limited ability of the methods to detect
mutations in the 'hot spots' and the other part of the RET
proto-oncogene or the other may result from the possibility that
RET proto-oncogene is not the only gene responsible for MEN 2A.
In fact, pheochromocytoma is present in -15% of patients with von
Rippel-Lindau disease (15,16) with few family members affected
with MTC (17). Further linkage analysis will be required to find
the genetic disorder of these patients.
Table 3. Genotype and aggressiveness of MTC
Genotype
C618S
4
M918T
Fisher exact test (two-tailed). N.S.: not significant.
~
2cm
p value*
Localized disease
Metastatic disease
6
8
<0.01
6
N.S.
0
C634R
C634Y
MTC
MTC< 2cm
Metastatic disease
593
Localized disease
2
0
N.S.
N.S.
594
Genotype and phenotype of MEN 2
A
B
6
E
~
E
4
0
l-
-
-o
o
(,)
2
0
Q)
N
en
(6348
•
• ~.
~
•
Qr:;O
4
I-
-
O·
:E
0
6
(6185
:E
0
2
Q)
N
en
0
0
0
40
20
60
80
D
C
•
6
-
6
•
o
E
~
()
o
~
0
20
- O·
en
:E
•
2
0
CD
60
2
N
.~
40
•
4
c
l-
••
f-
::i
en
M918T
C634Y
E 4
0
40
Age at operation for MTC (year)
Age at operation for MTC (year)
-s
20
0
80
60
40
20
0
80
60
80
Age at operation for MTC (year)
Age at operation for MTC (year)
Figure 1. Relationship between the tumor size or aggressiveness of MTC and age at operation in each genotype. Open circle, MTC confmed within the thyroid gland;
closed circle, MTC with lymph node metastases at the time of operation or persistent disease after radical operation confirmed by provocative test. (A) C618S;
(B) C634R; (C) C634Y; (D) M918T.
Table 4. Phenotype of pheochromocytoma in each genotype
Genotype
Uni-ibilateral
Serum catecholamine
Mono-:muIticentric lesion
Normal
C618S
1:1
N.!.
C618F
N.J.
N.!.
C618R
1:1
I:N.!.
4:10
1:11
Elevated ADR
Elevated NOR
Elevated ADR and NOR
C630S
C634R
C634G
1:0
0:1
C634Y
6:8
4:8
C634S
I: 1
N.!.:1
C634F
1:2
N.!.:1
C634W
N.!.
N.!.
M918T
1:0
1:0
No mutation
1:1 (bilateral ectopic)
I:1
N.!., not informative; ADR, adrenalin; NOR, noradrenalin.
5
9
4
9
2
Jpn J Clin OncoI1998;28(lO)
All genotypes developed MTC and the overall penetration of
MTC was more than 95%. Pheochromocytoma developed
equally in any genotype but not in C630S, which was found only
in the 'other' subtype. The frequency of pheochromocytoma was
not statistically different among various amino acid substitutions
at codon 618 or 634 (data not shown). Moers et al. (13)
investigated in a large family with a long-term and extensive
screening protocol, finding that C618 genotype develops pheochromocytoma, but less frequently than C634 genotype. They
suggested that MEN 2A families should not be subclassified into
MEN 2A and FMTC, but rather according to their specific
mutation in the RET proto-oncogene, like 'MEN 2A RET
C618S'. No malignant pheochromocytoma was found in the
present study. The laterality of the pheochromocytoma was not
statistically different among the genotypes. No RET point
mutation was found in the two patients who developed ectopic
pheochromocytoma and/or pheochromocytoma with an elevated
noradrenalin but not adrenalin level, suggesting that another
genetic disorder might be involved in tumorigenesis and catecholamine metabolism in these patients. Hyperparathyroidism developed in seven (12.3%) out of 49 patients with codon 634
mutations, but this frequency was not statistically different from
that in patients without codon 634 mutations. Since limited
screening in small families might bias the genotype-phenotype
correlation and will only reveal symptomatic patients with
manifest disease (preselection) and give an incomplete impression of the phenotype, the analysis for correlation between
specific genotype and the penetration of hyperparathyroidism
requires the collection of more specimens.
Different oncogenic transformations can be caused by different
mutations in the RET proto-oncogene (18). Mulligan and
co-workers (19,20) suggested that mutations of cysteine codon
618 or 620 lead to a milder course of disease compared with
mutations of codon 634. Moers et al. (13) described that the
clinical course of the disease in a family with C618 RET mutation
(n = 70) is mild compared with that for members with C634 RET
mutation (n = 115). In the present study, C618S genotype seems
to develop slow-growing MTC compared with those in other
genotypes with rapid growing MTC such as C634R, C634Y and
M918T (Fig. 1). On the other hand, two cases with C618R
genotype developed aggressive MTC (Table 2), i.e. a 39 y.o.
female had lymph node metastases although the primary tumor
was 1.2 em in diameter and a 44 y.o. female showed a positive
provocative calcitonin test after radical resection of MTC, 3 ern
in diameter, with no lymph node metastasis. Since there are only
two informative cases of C618R, the clinical aggressiveness of
MTC with C618R mutation cannot be defined without more
specimens of this genotype. The MTC in the patients with C634R
genotype hardly disseminated until the tumor grew larger than 2
ern in diameter, while the patients with C634Y had metastatic
disease in spite of small primary tumors (Fig. IB,C and Table 3).
The classical MEN 2B genotype, M918T, developed the most
aggressive and rapidly growing MTC (Fig. 1D).
595
CLINICAL IMPLICATIONS
Since conventional biochemical screening of MTC, pheochromocytoma and hyperparathyroidism may lead to false-negative
or false-positive results (21-23), the DNA testing should be
performed first to identify MEN 2 gene carriers. However, it is
still controversial when and on whom prophylactic thyroidectomy at an early age should be performed. Gill et al. (24) reported
cases of a 5 y 11m girl and her 3 y 8 m sister with C634R genotype
who developed metastatic MTC, suggesting the need for
prophylactic thyroidectomies in MEN 2A patients as young as 5
y.o. and strict yearly provocative screening beginning at 1 y.o.
Skinner et al. (25) recommended prophylactic thyroidectomy
with central neck lymph node dissection at 5 y.o. for patients with
MEN 2A, while they offer thyroidectomy in the first year of life
to MEN 2B gene carriers owing to the virulence and early onset
of MTC. In contrast, Lips et al. (7) emphasized that the risk of
complications of surgery (i.e. recurrent nerve paralysis and
hypoparathyroidism) in young children is not counterbalanced by
the gain that accrues from the prevention ofMTC, suggesting that
the surgery might be withheld until positive results of the
stimulation test or the age of 12-13 years, provided that periodic
examinations are conducted. Yamashita et al. (26) state that a
good result would be expected if the treatment is carried out at an
early stage, suggesting that thyroidectomy should be performed
when an elevation of calcitonin has been confirmed after positive
DNA testing.
The present study revealed that C618S mutation leads to
slow-growing MTC compared with C634 and M918 mutation
and that smaller MTC (1.2 em or less in diameter) of C634R
genotype still remains a local disease, while MTCs in C634Y can
easily spread in spite of a young age and small tumor. C634W and
M918T developed the most rapidly growingMTC at an early age.
These data suggest that prophylactic thyroidectomy for the
C634Y or M918T genotype carriers should be performed at an
early age, whereas it can be withheld in the C618S or C634R
carriers until the conventional biochemical or imaging examinations become positive. As for MEN 2A, we demonstrated that
the group of MEN 2A patients with C634Y mutation who need
early thyroidectomy can be distinguished from those with C618S
or C634R with regard to aggressiveness of the disease. Radical
lymph node dissection may be abbreviated for small MTC in
C618 or C634R (less than 1 em in diameter).
Concerning the penetration of pheochromocytoma, further
screening should be conducted for the FMTC and 'other'
kindreds whose genotypes are the same as that of MEN 2A.
Prophylactic adrenectomy is controversial because of its risk and
poor probability of penetration, laterality and multicentricity. In
order to obtain more correct genotype-phenotype correlations
and better clinical management, continued clinical evaluation of
patients and families in Japan is necessary.
596
Genotype and phenotype of MEN 2
Acknowledgments
The authors are indebted to the patients and families who
participated in this study for their cooperation, to Dr Kiyomi
Yamazaki of the Department of Endocrine Surgery, Tokyo
Women's Medical College, for contributing the nationwide
questionnaire survey, to Drs Koichi Nagasaki, Toshihiko Tsukada, Kazuki Sasaki and Kouji Maruyama of the Growth Factor
Division, National Cancer Center Research Institute, for fruitful
suggestions and to Ms Mon Ebinuma for her excellent technical
assistance. This research was supported in part by a Grant-in-Aid
from the Ministry of Health and Welfare for the Second-term
Comprehensive
10-Year
Strategy for Cancer Control and by
Grants-in-Aid for Cancer Research
(8-33
and
10-28)
from the
Ministry of Health and Welfare.
Affiliation of the Authors
IGrowth Factor Division, National Cancer Center Research
Institute, Tokyo, 2Department of Endocrine Surgery, Tokyo
Women's Medical University, Tokyo, 3Yaizu Municipal Hospital,
Yaizu, Shizuoka, 4Department of Internal Medicine, Gifu Red
Cross Hospital, Gifu, 5Kuma Hospital, Kobe, 6S e c ond Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Hokkaido, 7Department of Medicine III, Osaka University
Medical School, Suita, Osaka, 8Third Department of Internal
Medicine, Fukui Medical School,
Yoshida-gun, Fukui, 9S aitama
Cancer Center, Kitaadachi-gun, Saitama, 10Kitakyushu Municipal Medical Center, Kitakyushu, Fukuoka, 11Department of
Urology, Oita Medical University, Oita-gun, Oita, 12Se c o nd
Department of Surgery, Gunma University School of Medicine,
Maebashi, 13Department of Urology, Hamamatsu University
School of Medicine, Hamamatsu, Shizuoka, 14First Department
of Internal Medicine and 15Pirst Department of Surgery, University of Occupational and Environmental Health, Kitakyushu,
Fukuoka, 16Hyogo Prefecture Kaibara Hospital, Hikami-gun,
Hyogo, 17Se c ond Department of Surgery, School of Medicine,
University of Tokushima, Tokushima, 18Department of Medicine, Shinko Hospital, Kobe,
19Niigata Prefecture Central
Hospital, J oetsu, Niigata, 20Department of Surgery, Himeji Red
Cross Hospital, Himeji, and 21First Department of Internal
Medicine, Kobe University School of Medicine, Kobe, Japan
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