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

t-murakm@juntendo.ac.jp

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

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

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

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

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

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

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.

123

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

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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