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ORIGINAL ARTICLE
Clinicopathologic and immunohistochemical characteristicsof gastric adenocarcinoma with enteroblastic differentiation:a study of 29 cases
Takashi Murakami1,2 • Takashi Yao2 • Hiroyuki Mitomi3 • Takashi Morimoto1 •
Hiroya Ueyama1 • Kenshi Matsumoto1 • Tsuyoshi Saito2 • Taro Osada1 •
Akihito Nagahara1 • Sumio Watanabe1
Received: 21 December 2014 / Accepted: 1 April 2015 / Published online: 18 April 2015
� The International Gastric Cancer Association and The Japanese Gastric Cancer Association 2015
Abstract
Background Gastric adenocarcinoma with enteroblastic
differentiation (GAED) has been recognized as a variant of
alpha-fetoprotein (AFP)-producing gastric carcinoma,
although its clinicopathologic and immunohistochemical
features have not been fully elucidated.
Methods To elucidate the clinicopathologic and im-
munohistochemical features of GAED, we analyzed 29
cases of GAED, including ten early and 19 advanced lesions,
and compared these cases with 100 cases of conventional
gastric adenocarcinoma (CGA). Immunohistochemistry for
AFP, glypican 3, SALL4, and p53 was performed, and the
phenotypic expression of the tumors was evaluated by im-
munostaining with antibodies against MUC5AC, MUC6,
MUC2, CD10, and caudal-type homeobox 2 (CDX2).
Results Lymphatic and venous invasion was more fre-
quent in GAED (76 and 72 %) than in CGA (41 and 31 %;
P B 0.001). Lymph node metastasis was more frequently
observed in GAED (69 %) than in CGA (38 %;
P = 0.005), as were synchronous or metachronous liver
metastases (GAED, 31 %; CGA, 6 %; P B 0.001). Im-
munohistochemically, all GAED were positive for at least
one of three enteroblastic linage markers (AFP, glypican 3,
and SALL4). Glypican 3 was the most sensitive marker
(83 %) for GAED, followed by SALL4 (72 %) and AFP
(45 %), whereas no CGA was positive. Furthermore, the
rate of positive p53 staining was 59 % in GAED. Re-
garding the mucin phenotype, CD10 and CDX2 were dif-
fusely or focally expressed in all GAED cases. Invasive
areas with hepatoid or enteroblastic differentiation were
negative for CD10 and CDX2.
Conclusions Clinicopathologic features of GAED differ
from those of CGA. GAED shows aggressive biological
behavior, and is characteristically immunoreactive to AFP,
glypican 3, or SALL4.
Keywords Gastric adenocarcinoma with enteroblastic
differentiation � Alpha-fetoptotein-producing gastric
carcinoma � Glypican-3 � SALL4
Introduction
Gastric adenocarcinoma with enteroblastic differentiation
(GAED), also known as clear cell gastric carcinoma, is a
rare and not particularly well documented malignancy;
previous reports have been either small series or a single
case report [1, 2]. This type of tumor has been histo-
logically characterized as having a primitive intestine-like
structure, composed of cuboidal or columnar cells with
clear cytoplasm [1, 3, 4]. GAED has also been noted to
produce alpha-fetoprotein (AFP) in the serum and within
the tumor [1, 2], and it is recognized as a variant of AFP-
producing gastric carcinoma. However, the association
between GAED and AFP production remains unclear as
some GAED cases may be AFP negative [2, 5].
Since the first case of AFP-producing gastric cancer
with liver metastasis was reported in 1970 [6], many cases
& Takashi Murakami
[email protected]
1 Department of Gastroenterology, Juntendo University School
of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421,
Japan
2 Department of Human Pathology, Juntendo University
School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan
3 Department of Surgical and Molecular Pathology, Dokkyo
Medical University School of Medicine, Tochigi, Japan
123
Gastric Cancer (2016) 19:498–507
DOI 10.1007/s10120-015-0497-9
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of this tumor type have been reported. In previous reports,
AFP-producing gastric cancer was associated with a poor
prognosis, and advanced-stage disease usually presented
with liver metastases [7–10]. Histologically, typical AFP-
producing gastric cancer shows a hepatoid pattern com-
posed of neoplastic cells with abundant eosinophilic cyto-
plasm in solid nests [11], or a clear cell tubular pattern
resembling fetal gut epithelium [1, 3, 4]. Diagnosis of AFP-
producing gastric cancer is based on positive immunohis-
tochemical staining of AFP; however, some hepatoid or
enteroblastic tumors are negative for AFP expression.
Recent studies have demonstrated that glypican 3 and
SALL4 are also oncofetal proteins indicative of hepatocyte
differentiation [4, 12–16]. Glypican 3, a cell-surface hep-
aran sulfate proteoglycan, is present in fetal liver and
hepatocellular carcinoma or hepatoblastoma [4, 12–14].
Immunohistochemically, glypican 3-positive areas nearly
always overlap with AFP-positive areas in AFP-producing
gastric cancer [13]. SALL4 is a member of the SALL gene
family and acts as a zinc finger transcription factor. SALL4
has an essential role in maintaining the self-renewal and
pluripotency of embryonic stem cells [15, 16]. Accord-
ingly, glypican 3 and SALL4 have been suggested to be
sensitive markers for AFP-producing gastric cancer and
related tumors.
Thus, the aim of this study was to elucidate the
clinicopathologic and immunohistochemical features of
GAED in association with the immunostaining of AFP,
glypican 3, SALL4, and p53.
Materials and methods
Case selection
We studied 29 patients with GAED who underwent en-
doscopic or surgical resection at Juntendo University
Hospital, Tokyo, Japan, between January 2009 and March
2014. These included 10 early and 19 advanced lesions.
The diagnosis of GAED was based on the criteria described
by Matsunou et al. [1]: (1) columnar carcinoma cells grow
primarily in tubulopapillary and glandular patterns; (2)
carcinoma cells have clear cytoplasm and an oval nucleus
situated on the basal side; (3) abundant glycogen granules,
but no mucin, are contained in the clear cytoplasm.
Sex, age, tumor location, tumor size, macroscopic ap-
pearance, depth of invasion (pT stage), lymphatic or ve-
nous invasion, lymph node metastasis, hepatic metastasis,
and outcome were evaluated in all patients. The pT stage
was classified according to the seventh edition of the
American Joint Committee on Cancer/Union for Interna-
tional Cancer Control staging system [17]. We classified
pT1 tumors irrespective of lymph node or liver metastasis
as early lesions, and we defined the remaining tumors as
advanced lesions.
Immunohistochemistry
Serial tissue sections (4 mm thick) prepared from formalin-
fixed and paraffin-embedded tissues were subjected to
immunohistochemistry. For immunohistochemical ex-
amination, staining was performed using a Dako EnVision
kit with antibodies against AFP (rabbit polyclonal, 1:1000;
Dako, Glostrup, Denmark), glypican 3 (clone 1G12, 1:200;
BioMosaics, Burlington, VT, USA) SALL4 (clone 6E3,
1:100; Abnova, Taipei, Taiwan), and p53 (clone DO-7,
1:100; Dako, Glostrup, Denmark). The phenotypic ex-
pression of the tumors was evaluated by immunostaining
with antibodies against MUC5AC (NCL-MUC-5AC,
1:100; Novocastra, Newcastle-upon-Tyne, UK), MUC6
(NCL-MUC-6, 1:100; Novocastra, Newcastle upon Tyne,
UK), MUC2 (NCL-MUC-2, 1:100; Novocastra, Newcastle
upon Tyne, UK), CD10 (NCL-CD10-270, 1:100; Novo-
castra, Newcastle upon Tyne, UK), and caudal-type
homeobox 2 (CDX2; CDX2-88, 1:100; BioGenex, Fre-
mont, CA, USA). Appropriate positive and negative con-
trols were used for each antibody.
AFP, glypican 3, or SALL4 staining was assessed ac-
cording to a previous description [15]. Cytoplasmic stain-
ing for AFP, and membrane and cytoplasmic staining for
glypican 3 were evaluated. Only nuclear staining was
considered positive for SALL4. The scoring system was as
follows: score 0, less than 1 % of tumor cells positive;
score 1, 1–25 % of tumor cells positive; score 2, 26–50 %
of tumor cells positive; score 3, 51–75 % of tumor cells
positive; and score 4, more than 75 % of tumor cells
positive. The final results were reported as negative
(score 0) or positive (score 1, 2, 3, or 4).
The phenotypes were classified into four categories ac-
cording to the combination of the expression of CD10,
MUC2, MUC5AC, MUC6, and CDX2 [18]. Specimens
positive for MUC5AC or MUC6 were defined as gastric
type, those positive for MUC2, CD10, or CDX2 were de-
fined as intestinal type, and those with both phenotypes
were considered to be gastrointestinal type. In addition,
specimens with no CD10, MUC2, MUC5AC, or MUC6
expression were considered to be unclassified type. Ex-
pression of p53 was recorded as positive if distinct and
strong nuclear staining was observed in more than10 % of
tumor cells [19].
The histology and immunohistochemical staining results
were evaluated by two observers (T.M. and T.Y.). When
discrepancies arose, the cases were reviewed using a
multiheaded microscope to achieve a consensus.
Clinicopathologic and immunohistochemical characteristics of … 499
123
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Comparisons with conventional gastric
adenocarcinoma
We randomly selected 100 samples of conventional gastric
adenocarcinoma (CGA) from our files of endoscopically or
surgically resected specimens at our hospital between 2009
and 2014. These lesions were histologically papillary
adenocarcinomas, well to moderately differentiated tubular
adenocarcinomas, and poorly differentiated adenocarcino-
mas. We compared 29 GAED with 100 CGA in regard to
clinicopathologic findings such as sex, age, tumor location,
tumor size, macroscopic appearance, pT stage, lymphatic
or venous invasion, lymph node metastasis, and hepatic
metastasis. Furthermore, we randomly selected 20 lesions
from those 100 CGA, and performed immunohisto-
chemical staining using antibodies against AFP, glypi-
can 3, and SALL4 as described earlier.
Statistical analysis
All statistical analyses were performed using Stat-View for
Windows version 5.0 (SAS Institute, Cary, NC, USA).
Continuous data were compared with the Mann–Whitney
U test. Categorical analysis of variables was performed
using either the chi squared test (with Yates’s correction)
or Fisher’s exact test, as appropriate. A P value less than
0.05 was considered statistically significant.
Results
Clinicopathologic findings
The clinicopathologic findings of the 29 GAED patients
and the 100 CGA patients included in this study are sum-
marized in Table 1, and detailed clinicopathologic findings
of 29 GAED lesions are listed in Table 2. There were 23
male and six female GAED patients, with ages ranging
from 59 to 85 years (mean 73 years). Six GAED were
located in the upper area of the stomach, nine were in the
middle area, and 14 were in the lower area. The mean
tumor sizes of GAED were 19 mm for early lesions and
49 mm for advanced lesions (P\ 0.001). Macroscopically,
superficially depressed-type lesions were frequently ob-
served among early GAED cases, and types 2 and 3 were
frequently observed among advanced GAED cases,
similarly to CGA cases. With regard to the depth of in-
vasion, in the 29 GAED patients and the 100 CGA patients,
pT1 was observed in ten and 33 patients, respectively, pT2
was observed in four and 16 patients, respectively, pT3 was
observed in 12 and 37 patients, respectively, and pT4 was
observed in three and 14 patients, respectively. Neither of
these comparisons was considered statistically significant.
Lymphatic invasion was more frequently observed in
GAED (22 of 29 cases, 76 %) than in CGA (41 of 100
cases, 41 %; P = 0.001), as was venous invasion (GAED,
72 %; CGA, 31 %; P B 0.001). Lymphatic and/or venous
invasion was present in 26 GAED (90 %), including eight
early lesions and 18 advanced lesions. Lymph node
metastasis was more frequently observed in GAED (20 of
29 cases, 69 %), including four early cancer patients and
Table 1 Clinicopathologic features of gastric adenocarcinoma
studied
Adenocarcinoma with
enteroblastic differentiation
(n = 29)
Conventional
adenocarcinoma
(n = 100)
Sex
Male 23 (79 %) 74 (74 %)
Female 6 (21 %) 26 (26 %)
Age (years) 73.0 ± 7.5 70.6 ± 7.8
Location of tumor
Upper 6 (21 %) 26 (26 %)
Middle 9 (31 %) 31 (31 %)
Lower 14 (48 %) 43 (43 %)
Size of tumor (mm) 38.6 ± 23.2 39.6 ± 19.6
Macroscopic type
Superficial 10 (35 %) 33 (33 %)
Type 1 1 (3 %) 6 (6 %)
Type 2 10 (35 %) 29 (29 %)
Type 3 7 (24 %) 26 (26 %)
Type 4 0 (0 %) 5 (5 %)
Unclassified 1 (3 %) 1 (1 %)
Deptha
pT1 10 (35 %) 33 (33 %)
pT2 4 (14 %) 16 (16 %)
pT3 12 (41 %) 37 (37 %)
pT4 3 (10 %) 14 (14 %)
Lymphatic invasion
Yes 22 (76 %) 41 (41 %)
No 7 (24 %) 59 (59 %)
Venous invasion
Yes 21 (72 %) 31 (31 %)
No 8 (28 %) 69 (69 %)
Lymph nodes metastasis
Yes 20 (69 %) 38 (38 %)
No 9 (31 %) 62 (62 %)
Liver metastasis
Yes 9 (31 %) 6 (6 %)
No 20 (69 %) 94 (94 %)
Age and tumor size are presented as the mean ± standard deviation.a Depth of tumor invasion (pT stage) was classified according to the
American Joint Committee on Cancer/Union for International Cancer
Control staging system (seventh edition)
500 T. Murakami et al.
123
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Table
2Summaryofclinicopathologic
featuresandhistologic
findingsin
each
case
ofadenocarcinomawithenteroblastic
differentiation
Case
Sex
Age
Size
Location
Depth
aLymphatic
invasion
Venous
invasion
Lymphnodes
metastasis
Liver
metastasis
Histologic
findings
Percentageof
enteroblastic
differentiation
component(%
)
Outcome
1M
78
14
Upper
pT1
-?
--
Entandcon(w
ell)
60
13months,ANED
2M
75
18
Upper
pT1
-?
--
Entandcon(w
ell)
40
26months,ANED
3M
74
21
Middle
pT1
--
--
Entandcon(m
od)
30
34months,ANED
4M
77
11
Middle
pT1
-?
--
Entandcon(w
ell)
50
30months,ANED
5M
83
36
Middle
pT1
??
?-
Entandcon
(wellandmod)
50
16months,ANED
6F
70
40
Lower
pT1
?-
??
Ent,con
(wellandmod)andhep
60
17months,DOD
7M
61
8Lower
pT1
--
--
Entandcon(w
ell)
40
39months,ANED
8M
71
16
Lower
pT1
??
??
Entandcon
(well,mod,andpoor)
80
28months,DOD
9M
82
20
Lower
pT1
?-
?-
Entandcon
(wellandmod)
60
20months,ANED
10
F80
6Lower
pT1
?-
--
Entandcon
(wellandmod)
50
2months,unknown
11
M74
60
Upper
pT2
-?
--
Ent,con
(wellandmod)andhep
70
27months,ANED
12
M74
25
Middle
pT2
??
??
Entandcon
(wellandmod)
40
3months,DOD
13
M74
90
Lower
pT2
??
??
Entandcon
(wellandpoor)
80
2months,DOD
14
F70
25
Lower
pT2
??
?-
Entandcon
(wellandmod)
80
24months,ANED
15
M70
30
Upper
pT3
??
?-
Entandcon
(modandpoor)
60
30months,DOD
16
M65
30
Upper
pT3
??
?-
Entandcon
(wellandmod)
80
25months,ANED
17
M83
21
Upper
pT3
??
??
Entandcon
(modandpoor)
70
19months,DOD
18
F59
44
Middle
pT3
??
??
Entandcon(m
od)
60
6months,DOD
19
M63
75
Middle
pT3
--
--
Entandcon
(wellandpoor)
70
26months,ANED
20
M70
30
Middle
pT3
??
?-
Entandcon
(wellandmod)
80
18months,ANED
21
M66
60
Middle
pT3
??
?-
Entandcon
(wellandmod)
70
9months,DOD
Clinicopathologic and immunohistochemical characteristics of … 501
123
Page 5
Table
2continued
Case
Sex
Age
Size
Location
Depth
aLymphatic
invasion
Venous
invasion
Lymphnodes
metastasis
Liver
metastasis
Histologic
findings
Percentageof
enteroblastic
differentiation
component(%
)
Outcome
22
M68
44
Lower
pT3
??
??
Ent,con(m
od)andhep
30
12months,DOD
23
F85
30
Lower
pT3
??
--
Entandcon
(wellandmod)
90
23months,DOD
24
M60
75
Lower
pT3
??
??
Entandcon
(modandpoor)
60
11months,DOD
25
M72
72
Lower
pT3
??
?-
Entandcon
(wellandmod)
90
14months,DOD
26
M73
46
Lower
pT3
?-
?-
Entandcon
(well,mod,andpoor)
80
12months,DOD
27
M85
38
Middle
pT4
??
?-
Ent,con(m
od)andylk
30
6months,DOD
28
M84
65
Lower
pT4
??
??
Entandcon
(wellandmod)
70
28months,DOD
29
F72
70
Lower
pT4
?-
?-
Entandcon
(modandpoor)
70
13months,DOD
Mmale,Ffemale,Upper
upper
thirdofstomach,Middle
middle
thirdofstomach,Lower
lower
thirdofstomach,Entgastric
adenocarcinomawithenteroblastic
differentiation,Hep
hepatoid
adenocarcinoma,
Ylk
yolk
sactumorlikecarcinoma,
Conconventional
adenocarcinoma,
wellwell-differentiated
typeofconventional
adenocarcinoma,
modmoderately-differentiated
typeof
conventional
adenocarcinoma,
poorpoorly-differentiated
typeofconventional
adenocarcinoma,
ANED
alivewithnoevidence
ofdisease,DOD
diedofdisease,plussignpresent,minussign
absent
aDepth
oftumorinvasion(pTstage)
was
classified
accordingto
theAmerican
JointCommitteeonCancer/UnionforInternational
CancerControlstagingsystem
(seventh
edition)
502 T. Murakami et al.
123
Page 6
16 advanced cancer patients, than in CGA (38 of 100 cases,
38 %; P = 0.005. Synchronous or metachronous liver
metastases were more frequently found in GAED (nine of
29 cases, 31 %) than in CGA (six of 100 cases, 6 %;
P B 0.001).
Histopathologic findings
The histopathologic findings of the 29 GAED specimens
are summarized in Table 2. All 29 cases showed adeno-
carcinoma composed of cuboidal or columnar cells with
clear cytoplasm, resembling the primitive gut (Fig. 1a). In
cases 6, 11, and 22, part of the invasive area exhibited a
hepatoid pattern (Fig. 1b); in case 27, the tumor partly
exhibited a yolk sac tumor pattern, representing reticular or
papillary structures composed of cuboidal or columnar
cells (Fig. 1c). Enteroblastic adenocarcinoma was com-
bined with conventional well-differentiated or moderately-
differentiated tubular adenocarcinoma in the upper part of
all tumors (Fig. 1d).
Outcomes
The duration of follow-up ranged from 2 to 39 months
(mean, 18.4 months; Table 2). Patient 10 was lost to fol-
low-up 2 months after endoscopic therapy. Complete fol-
low-up clinical data were obtained from the remaining 28
GAED patients. Fifteen patients, including two early can-
cer patients and 13 advanced cancer patients, died as a
result of their disease at an average of 14.6 months after
surgery. Moreover, all nine patients with liver metastasis
died within 3 years of surgery.
Immunohistochemical findings
The results of the immunohistochemical analyses are
summarized in Table 3. All GAED were positive for at
least one of three enteroblastic linage markers (AFP, gly-
pican 3, and SALL4; Fig. 2). Glypican 3 was the most
sensitive marker (positivity, 83 %), followed by SALL4
(72 %) and AFP (45 %). Diffuse marker positivity (score
2, 3, or 4) was more frequently identified in advanced le-
sions (AFP, 26 %; glypican 3, 47 %; SALL4, 58 %) than
in early lesions (AFP, 10 %; glypican 3, 20 %; SALL4,
30 %). By contrast, there was no expression of AFP, gly-
pican 3, and SALL4 in 20 cases of CGA. In addition, the
rate of positive p53 staining was 59 % in all GAED lesions.
With regard to phenotypic expression, 15 GAED cases
(52 %) exhibited the pure intestinal phenotype, whereas the
remaining 14 cases (48 %) exhibited the gastrointestinal
phenotype. CD10 and CDX2 were diffusely or focally
expressed in all GAED cases (Fig. 2d).
Association of marker expression and tumor
invasion, metastases, or outcomes
In GAED cases, we further analyzed the associations of
mucin phenotype and expression ofAFP, glypican 3, SALL4,
and p53 with venous invasion, lymphatic invasion, lymph
nodemetastasis, hepatic metastasis, and outcome. There were
Fig. 1 Histologic features of
gastric adenocarcinoma with
enteroblastic differentiation.
a A major portion of the lesion
in case 16 was composed of
cuboidal or columnar cells with
clear cytoplasm resembling the
primitive gut. b A minor portion
of the lesion in case 11
exhibited hepatoid
adenocarcinoma. c In case 27,
the tumor partly exhibited a
yolk sac tumor pattern,
representing reticular or
papillary structures composed
of cuboidal or columnar cells.
d Enteroblastic adenocarcinoma
was combined with
conventional well-differentiated
or moderately-differentiated
tubular adenocarcinoma in the
upper part of the tumor in case 9
Clinicopathologic and immunohistochemical characteristics of … 503
123
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no significant associations, although glypican 3 and SALL4
expression tended to be associated with lymphatic invasion
(glypican 3, P = 0.069; SALL4, P = 0.074).
Discussion
In this study, patients with GAED were predominantly
male (79 %), and GAED lesions were frequently located in
the middle third and lower third of the stomach (79 %),
similarly to CGA [20–22]. The mean age of patients at
diagnosis tended to be older than that of patients with CGA
[20–22]. Lymphatic and vascular involvements are com-
mon among AFP-producing gastric carcinomas [10]. The
prevalence of lymphatic involvement and that of venous
involvement in GAED (76 and 72 %, respectively) were
remarkably higher than those in CGA (10–56 %) [21, 23].
In this study, most GAED patients had lymphatic and/or
vascular invasion (90 %). Lymph node metastasis was
detected in 40 % of early lesions and 84 % of advanced
lesions, and these rates are also higher than those associ-
ated with CGA (26–45 %) [20, 21]. Furthermore, 31 % of
GAED patients had synchronous or metachronous liver
metastasis. Our findings were in accordance with a review
of 270 patients with AFP-producing gastric carcinoma,
which reported an 83 % incidence of lymph node metas-
tasis and a 33 % incidence of liver metastasis [10]. AFP-
producing gastric cancers are typically associated with a
poor prognosis and high rates of liver metastasis [9].
Hence, our data suggest that GAED, as well as AFP-pro-
ducing gastric cancer, is associated with a poor prognosis.
With the exception of hepatocellular carcinoma or yolk
sac tumors, gastric cancer is one of the commonest AFP-
producing tumors [4, 6–10, 24–27]. Gastric hepatoid ade-
nocarcinoma or GAED is one of the representative histo-
logic types of AFP-producing gastric cancer. AFP
production is generally considered the result of retrodif-
ferentiation of tumor cells into fetal cells capable of pro-
ducing AFP [1, 28]; therefore, this suggests that gastric
hepatoid adenocarcinoma and GAED exhibit retrodiffer-
entiation. However, some hepatoid tumors are negative for
AFP expression, and do not have the ability to produce
AFP. Similarly, the rate of negative AFP staining was 55 %
in GAED, suggesting that some GAED do not have the
ability to produce AFP. The histogenesis of AFP-producing
gastric carcinoma still remains unclear. AFP-producing
gastric carcinoma is thought to develop from conventional
tubular adenocarcinoma, subsequently progressing to
GAED with hepatoid differentiation and AFP production
[4]. Accordingly, we also found that GAED lesions were
mixed with conventional well-differentiated or moderately-
differentiated tubular adenocarcinoma in all cases.
Recent studies have demonstrated that SALL4 is
specifically expressed not only in primitive germ cell tu-
mors but also in gastric carcinoma with fetal gut differ-
entiation [15, 16]. In our study, expression of SALL4 was
positive in 20 cases (72 %), which was higher than AFP
positivity (45 %), indicating that SALL4 was a more sen-
sitive marker of fetal gut differentiation than AFP. Ushiku
et al. [15] stated that SALL4 is a sensitive marker for AFP-
producing gastric carcinoma and is especially useful to
diagnose hepatoid gastric carcinoma. The previous report
and our findings suggest that SALL4 is useful to diagnose
Table 3 Immunohistochemical characteristics of adenocarcinoma
with enteroblastic differentiation
Adenocarcinoma
with enteroblastic
differentiation (n = 29)
AFP
Score 0 16 (55 %)
Score 1 7 (24 %)
Score 2 4 (14 %)
Score 3 2 (7 %)
Score 4 0 (0 %)
Glypican 3
Score 0 5 (17 %)
Score 1 13 (45 %)
Score 2 3 (10 %)
Score 3 4 (14 %)
Score 4 4 (14 %)
SALL4
Score 0 8 (28 %)
Score 1 7 (24 %)
Score 2 5 (17 %)
Score 3 4 (14 %)
Score 4 5 (17 %)
p53a
Negative 12 (41 %)
Positive 17 (59 %)
CDX2b
Focal 12 (41 %)
Diffuse 17 (59 %)
Phenotype
Gastrointestinal 14 (48 %)
Intestinal 15 (52 %)
The scoring system was as follows: score 0, less than 1 % of tumor
cells positive; score 1, 1–25 % of tumor cells positive; score 2,
26–50 % of tumor cells positive; score 3, 51–75 % of tumor cells
positive; and score 4, more than 75 % of tumor cells positive
AFP alpha-fetoprotein, CDX2 caudal-type homeobox 2a p53 positivity was recorded if distinct and strong nuclear staining
was observed in more than 10 % of the tumor cellsb CDX2 positivity of tumors was classified as follows: less than 1 %,
negative; 1–30 %, focal; more than 30 %, diffuse
504 T. Murakami et al.
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not only hepatoid gastric carcinoma but also a series of
variants of gastric carcinoma with retrodifferentiation such
as GAED. Glypican 3 is also a sensitive marker of hepatoid
components of AFP-producing gastric carcinoma [4, 12–
14]. In the present study, glypican 3 expression was ob-
served in 24 cases (83 %), and was thereby the most sen-
sitive marker for GAED. The Zhx2 gene, the mouse
orthologue of human ZHX2, acts as a repressor of both AFP
and glypican 3 expression in the adult mouse liver [29, 30],
and it is often silenced in hepatocellular carcinoma in as-
sociation with Zhx2 promoter hypermethylation. In addi-
tion, ZHX2 mRNA levels were decreased in hepatocellular
carcinoma cases with high serum AFP levels [31]. It is of
further interest to investigate the relationship between
ZHX2 repression and glypican 3 or AFP expression in
GAED.
Protein p53 plays a crucial role in cell apoptosis. Wild-
type p53 induces growth arrest at the G1/S phase of the cell
cycle in response to DNA damage, thus inhibiting the
proliferation of cells [32]. Mutated p53 loses this inhibitive
function, thus allowing cells with damaged DNA to pro-
liferate. In this study, we found that p53 expression did not
correlate with clinicopathologic parameters [33]. It is
controversial whether p53 expression predicts prognosis;
however, some studies have reported that p53 has no as-
sociation with prognosis in conventional gastric cancer or
AFP-producing gastric cancer patients [34–36], a finding
similar to that in our study.
With regard to mucin phenotype, most AFP-producing
gastric carcinomas are classified as the intestinal type [4,
37], in accordance with our study. Invasive areas with
hepatoid or enteroblastic differentiation were negative for
CD10 and CDX2 expression, suggesting the retrodiffer-
entiation of tumor cells (Fig. 2e, f).
In conclusion, GAED, resembling the primitive gut,
showed aggressive behavior such as lymphatic and venous
invasion, lymph node metastasis, and liver metastasis, and
its clinicopathologic features were similar to those of AFP-
producing gastric carcinoma but were not similar to those
of CGA. In addition, GAED was immunoreactive to AFP,
Fig. 2 Immunohistochemical
staining of gastric
adenocarcinoma with
enteroblastic differentiation.
Positive findings for alpha-
fetoprotein in case 11 (a), forglypican 3 in case 23 (b), forSALL4 in case 11 (c), and for
caudal-type homeobox 2 in case
25 (d). In case 11, the tumor
exhibited an enteroblastic
structure (e, left) accompanied
by a hepatoid structure (e,right). f The immunostaining of
CD10 in the same area as that
shown in e. CD10 was
expressed preferentially in the
enteroblastic area (f, left), butnot in the hepatoid area (f, right)
Clinicopathologic and immunohistochemical characteristics of … 505
123
Page 9
glypican 3, or SALL4. These findings indicate that some
GAED have the ability to produce AFP and overlap with
AFP-producing gastric carcinoma.
Acknowledgments We thank Noriko Sasahara and Isao Kura-
hayashi (Department of Human Pathology, Juntendo University
School of Medicine) for their assistance with the histologic analysis.
Conflict of interest The authors declare no conflict of interest.
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