Arch Pathol Lab MedVol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass LesionsWang et al 1723

Original Articles
Glypican-3 as a Useful Diagnostic Marker That
Distinguishes Hepatocellular Carcinoma From Benign
Hepatocellular Mass Lesions
Hanlin L. Wang, MD, PhD; Florencia Anatelli, MD; Qihui Jim Zhai, MD; Brian Adley, MD;
Shang-Tian Chuang, DO; Ximing J. Yang, MD, PhD
Context.Histopathologic distinction between hepato-
cellular carcinoma (HCC) and benign hepatocellular mass
lesions, particularly hepatocellular adenoma, can some-
times be challenging. The currently available ancillary tools
are suboptimal in terms of sensitivity and specicity.
Objective.To further characterize the diagnostic value
of glypican-3 (GPC3), a cell surface proteoglycan that has
recently been shown to be overexpressed in HCC, in the
distinction between HCC and benign hepatocellular mass
lesions.
Design.A total of 221 surgically resected liver speci-
mens were subjected to immunohistochemical staining us-
ing a monoclonal antibody specic for GPC3. These in-
cluded 111 HCCs, 48 hepatocellular adenomas, 30 focal
nodular hyperplasias, and 32 large regenerative nodules in
the background of cirrhosis.
Results.Cytoplasmic, membranous, and canalicular
staining for GPC3 was detected in 84 (75.7%) of the 111
HCCs, among which, 61 (72.6%) of the 84 cases exhibited
diffuse immunoreactivity. In contrast, none of the 110 cas-
es of hepatocellular adenoma, focal nodular hyperplasia,
and large regenerative nodule showed detectable GPC3
staining. Focal GPC3 immunoreactivity was detected in
cirrhotic nodules in 11 (16.4%) of 67 HCC cases with a
cirrhotic background, but no background staining was ob-
served in the remaining 44 HCCs without cirrhosis. GPC3
expression in HCCs did not correlate with the size, differ-
entiation, or stage of the tumors; the presence or absence
of cirrhotic background; or the underlying etiologies.
Conclusions.GPC3 is a specic immunomarker for
HCC that can be used to distinguish HCC from benign he-
patocellular mass lesions, particularly hepatocellular ade-
noma. However, the diagnosis of HCC should not rely en-
tirely on positive GPC3 immunostaining because focal im-
munoreactivity can be detected in a small subset of cir-
rhotic nodules. In addition, GPC3 expression in HCC can
also be focal, and thus, the lack of GPC3 staining does not
exclude the diagnosis of HCC.
(Arch Pathol Lab Med. 2008;132:17231728)
G
lypican-3 (GPC3), a member of the heparan sulfate
proteoglycan family, is an oncofetal protein that is
expressed in the embryo and involved in morphogenesis
and growth control during development.
1,2
Its expression
is silenced in adult tissues, and loss-of-function mutations
are responsible for Simpson-Golabi-Behmel syndrome, a
rare X-linked prenatal and postnatal overgrowth with
multiple congenital anomalies and increased risk of neo-
Accepted for publication April 14, 2008.
From the Department of Pathology and Laboratory Medicine, Cedars-
Sinai Medical Center, Los Angeles, Calif (Dr Wang); the Department of
Pathology and Immunology, Washington University School of Medi-
cine, St Louis, Mo (Dr Anatelli); the Department of Pathology, Weill
Medical College of Cornell University, The Methodist Hospital, Hous-
ton, Tex (Dr Zhai); and the Department of Pathology, Northwestern
University Feinberg School of Medicine, Chicago, Ill (Drs Adley,
Chuang, and Yang).
The authors have no relevant nancial interest in the products or
companies described in this article.
Presented in part at the 96th Annual Meeting of the United States
and Canadian International Academy of Pathology, San Diego, Calif,
March 2430, 2007.
Reprints: Hanlin L. Wang, MD, PhD, Department of Pathology and
Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Blvd,
Los Angeles, CA 90048 (e-mail: hanlin.wang@cshs.org).
plasias in infancy.
3,4
In vitro studies have shown that
GPC3 induces apoptosis in certain cell lines via the an-
choring of the protein to the cell membrane,
5
indicating
that GPC3 may function as an inhibitor of cell proliferation
and a tumor suppressor in a cell linespecic manner.
Glypican-3 as a potential tumor marker for hepatocel-
lular carcinoma (HCC) was rst suggested in 1997 by Hsu
et al,
6
who reported that GPC3 mRNA was preferentially
expressed in HCCs. In their study of primary and/or re-
current HCCs from 154 patients, GPC3 (named MXR7 in
their study) mRNA was detected in 143 (74.8%) of the 191
tumors, in contrast to only 3 cirrhotic and 2 (3.2%) of the
noncirrhotic adult livers that showed low levels of GPC3
mRNA expression. In addition, GPC3 mRNA was not de-
tected in 3 hepatocellular adenomas included in their
study. These observations were subsequently conrmed
by Zhu et al
7
in a similar study.
In 2003, several studies showed increased GPC3 ex-
pression at the protein level in HCC, when compared with
healthy livers and benign hepatic lesions.
811
In the study
by Capurro et al,
8
GPC3 protein expression was demon-
strated by immunohistochemistry in 21 (72%) of 29 HCC
cases but was not detected in healthy livers or various
benign liver lesions, including 22 cirrhotic livers, 7 hepa-
1724 Arch Pathol Lab MedVol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass LesionsWang et al
tocellular adenomas, and 4 focal nodular hyperplasias. In
addition, GPC3 can be detected in the serum as a secreted
protein in a subset of patients with HCC but is undetect-
able in healthy individuals or patients with hepatitis or
cirrhosis.
8,10,12,13
Interestingly, elevated serum GPC3 levels
in most HCC patients do not correlate with their serum
-fetoprotein values, and GPC3 appears to be more sen-
sitive than -fetoprotein as a serologic marker. Therefore,
simultaneous use of both GPC3 and -fetoprotein would
improve overall sensitivity in HCC detection.
One of the challenges in histopathologic diagnosis of
hepatocellular mass lesions is to distinguish HCC (partic-
ularly well differentiated) from hepatocellular adenoma,
and the available immunomarkers in clinical practice, such
as -fetoprotein, polyclonal carcinoembryonic antigen,
and CD34, have signicant diagnostic limitations.
14,15
In
this regard, GPC3 has shown promise as a useful diag-
nostic immunomarker as demonstrated in 3 studies in
which a total of 28 hepatocellular adenomas were immu-
nohistochemically examined.
8,16,17
In the current study, a
large number of hepatocellular adenomas and other be-
nign hepatocellular mass lesions were immunohistochem-
ically analyzed for GPC3 expression to further character-
ize its diagnostic value in distinguishing them from HCC.
MATERIALS AND METHODS
A total of 221 liver resection specimens were included in this
study. These included 111 HCCs, 48 hepatocellular adenomas, 30
focal nodular hyperplasias, and 32 large regenerative nodules
arising in cirrhotic livers. These cases were retrieved from sur-
gical pathology archives of Washington University BarnesJewish
Hospital in St Louis, Mo; The Methodist Hospital in Houston,
Tex; and Northwestern University Memorial Hospital in Chicago,
Ill. The clinical history, pathology reports, and slides stained with
hematoxylin-eosin were reviewed to conrm the diagnoses and
to determine the underlying etiologies, when applicable. Hepa-
tocellular carcinoma cases, in which the patients had undergone
preoperative chemoembolization with tumor necrosis, were ex-
cluded from this study.
Immunohistochemical staining for GPC3 was performed fol-
lowing the protocol described previously.
18
Briey, 4-m sections
from formalin-xed, parafn-embedded tissue blocks were de-
parafnized, rehydrated, and treated with 3% hydrogen peroxide
for 15 minutes to inhibit endogenous peroxidase. Following mi-
crowave heat-induced epitope retrieval in 0.1M citrate buffer at
pH 6.0 for 20 minutes, the slides were incubated with a mouse
monoclonal antibody specic for GPC3 (clone 1G12) obtained
from BioMosaics (Burlington, Vt), with a dilution of 1:200 for 1
hour at room temperature. After incubation with a rabbit, anti-
mouse secondary antibody, a reaction was performed using the
EnVision detection system that contains biotin-free horseradish
peroxidaselabeled polymers obtained from Dako Corporation
(Carpinteria, Calif). The staining was visualized using 3,3-dia-
minobenzidine substratechromogen solution and counterstained
with hematoxylin.
Immunohistochemically stained slides were independently
evaluated by 3 observers (H.L.W., F.A., and X.J.Y.). Cases with
signicantly discrepant interpretation were resolved by rereview
together by 2 of the observers (H.L.W. and F.A.). A case was
considered negative if 5% of the cells of interest exhibited im-
munoreactivity. Positive stains were further stratied as focal (5%
to 50% of the cells stained) and diffuse (50% of the cells
stained). Different staining patterns (cytoplasmic, membranous,
or canalicular) were recorded.
Statistical analysis was performed using the 2-tailed Fisher ex-
act test or the
2
test with the Yates continuity correction. A P
value of .05 was considered statistically signicant.
RESULTS
Clinicopathologic Features of Hepatocellular Lesions
The ages of patients with HCC ranged from 26 to 82
years (mean, 60.1 years; median: 62 years). Of the 111 pa-
tients, 70 were men, and 41 were women, for a male to
female ratio of 1.71:1. At the time of surgical resection,
tumors ranged in size from 0.6 to 18.0 cm (mean, 5.2 cm;
median, 4.0 cm). Tumors in 42 (37.8%) of the 111 cases
were stage I, 42 (37.8%) stage II, 19 (17.1%) stage IIIA, 2
(1.8%) stage IIIB, and 6 (5.4%) stage IV. Histopathologi-
cally, 51 (46%) of the HCC tumors were well differentiat-
ed, 45 (40.5%) were moderately differentiated, and 15
(13.5%) were poorly differentiated. Sixty-seven (60.4%) of
the tumors were detected in a cirrhotic background,
whereas 44 (39.6%) arose in noncirrhotic livers. The un-
derlying etiologies associated with HCC development
could not be determined in 33 cases (29.7%), among
which, 78.8% (26/33) of the HCCs arose in noncirrhotic
livers. In the remaining cases, hepatitis C was documented
in 42 (37.8%) cases, hepatitis B in 12 cases (10.8%), hepa-
titis B and C coinfection in 2 cases (1.8%), hepatitis B and
C coinfection plus excessive alcohol use in 6 cases (5.4%),
excessive alcohol use in 9 cases (8.1%), possible nonalco-
holic steatohepatitis in 5 cases (4.5%), familial hemochro-
matosis in 1 case (0.9%), and
1
-antitrypsin deciency in
1 case (0.9%).
The ages of patients with hepatocellular adenoma
ranged from 10 to 83 years (mean, 36.8 years; median, 33
years). Of the 48 patients, 43 (89.6%) were women and 5
(10.4%) were men, for a female to male ratio of 8.6:1. Elev-
en (25.6%) of the female patients had a documented his-
tory of using oral contraceptives. At the time of surgical
resection, the tumors ranged in size from 0.6 to 25.0 cm
(mean, 6.4 cm; median, 6.1 cm). Four (8.3%) of the 48 pa-
tients had more than 1 tumor.
The ages of patients with focal nodular hyperplasia
ranged from 9 to 78 years (mean, 37.2 years; median, 38
years). Of the 30 patients, 28 (93.3%) were women and 2
(6.7%) were men, for a female to male ratio of 14:1. Two
(7.1%) of the female patients had a documented history of
using oral contraceptives. At the time of surgical resection,
the lesions ranged in size from 0.3 to 14.0 cm (mean, 4.3
cm; median, 4.3 cm).
The ages of patients with large, regenerative nodules in
a cirrhotic background ranged from 30 to 69 years (mean,
55.6 years; median, 58 years). Of the 32 patients, 21
(65.6%) were men and 11 (34.4%) were women, for a male
to female ratio of 1.9:1. The underlying etiologies for cir-
rhosis included hepatitis C (14/32; 43.8%), hepatitis B (2/
32; 6.3%), hepatitis C and B coinfection (1/32; 3.1%), ex-
cessive alcohol use (3/32; 9.4%),
1
-antitrypsin deciency
(2/32; 6.3%), primary sclerosing cholangitis (1/32; 3.1%),
and cryptogenic origin (9/32; 28.1%).
Immunohistochemical Findings of GPC3 Expression in
Hepatocellular Lesions
As shown in Table 1, positive immunostaining for GPC3
was observed in 84 (75.7%) of the 111 HCC cases, among
which, 61 (72.6%) cases showed diffuse immunoreactivity.
In marked contrast, none of the 110 benign hepatocellular
mass lesions, including 48 hepatocellular adenomas, 30 fo-
cal nodular hyperplasias and 32 large regenerative nod-
ules, showed GPC3 immunoreactivity.
In GPC3-expressing HCCs, 3 different immunostaining
Arch Pathol Lab MedVol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass LesionsWang et al 1725
Table 1. Summary of Immunohistochemical Findings
of Glypican-3 Expression in Hepatocellular
Mass Lesions*
Immunoreactivity
(%)
HCC,
No. (%)
(n 111)
HCA,
No. (%)
(n 48)
FNH,
No. (%)
(n 30)
LRN,
No. (%)
(n 32)
Negative (5) 27 (24.3) 48 (100) 30 (100) 32 (100)
Focal (550) 23 (20.7) 0 0 0
Diffuse (50) 61 (55.0) 0 0 0
* HCC indicates hepatocellular carcinoma; HCA, hepatocellular ad-
enoma; FNH, focal nodular hyperplasia; and LRN, large regenerative
nodule.
Figure 1. Examples of different glypican-3 immunostaining patterns
observed in hepatocellular carcinomas: (A) predominantly membra-
nous, (B) predominantly canalicular, and (C) predominantly cytoplas-
mic (original magnications 400).
patterns were observed: predominantly membranous (Fig-
ure 1, A), predominantly canalicular (Figure 1, B), and
predominantly cytoplasmic (Figure 1, C). These different
staining patterns did not appear to correlate with the dif-
ferentiation status or the growth pattern of the tumors. In
fact, more than one third of the tumors exhibited a mixed
staining pattern within the same tumors, although the tu-
mor cells with different staining patterns showed similar
or identical histologic features (Figure 2, A). Similarly, in
those tumors that were focally positive for GPC3 expres-
sion, the tumor cells showing negative immunoreactivity
did not appear to be dissimilar morphologically to those
with positive staining within the same tumors (Figure 2,
B).
Table 2 further demonstrates that GPC3 immunoreactiv-
ity detected in HCCs did not correlate with the differen-
tiation status of the tumors. Although poorly differenti-
ated tumors tended to express GPC3 more frequently than
well-differentiated HCCs (93.3% vs 66.7%), the difference
did not reach a statistical signicance (P .05). In addi-
tion, HCCs arising in cirrhotic livers expressed GPC3 at a
similar frequency (76.1%) and with similar staining pat-
terns (Figure 3, A) to those without a cirrhotic background
(75%; Figure 3, B). Furthermore, no correlation was ob-
served between GPC3 expression and the size or stage of
the tumors or their underlying etiologies.
With respect to various morphologic variants, 5 (55.6%)
of the 9 clear cell HCCs and 4 (80%) of 5 pelioid HCCs
exhibited GPC3 immunoreactivity. There was only 1 case
of sarcomatoid HCC included in our study, which showed
focal GPC3 expression. There was also only 1 case of -
brolamellar HCC, which showed a diffuse staining pat-
tern.
Among 67 HCCs with a cirrhotic background, focal
GPC3 immunoreactivity was detected in a small propor-
tion of the cirrhotic nodules in 11 (16.4%) cases. The stain-
ing typically involved periseptal hepatocytes, which were
morphologically indistinguishable from the hepatocytes
that did not stain for GPC3, either in the same nodules or
in different nodules (Figure 4). The HCCs in these 11 cases
were all positive for GPC3 expression, 7 (63.6%) diffuse
and 4 (36.4%) focal, typically with a stronger staining in-
tensity relative to that detected in cirrhotic nodules. No
background staining was observed in nonneoplastic he-
patocytes in any of the 44 HCC cases without cirrhosis.
COMMENT
HCC is the fth most common cancer and the third
most common cause of cancer-related deaths worldwide.
19
Accurate diagnosis is critically important to appropriate
clinical management of the patients and assessment of the
prognosis. Although histopathologic diagnosis of HCC
may be straightforward in many cases, challenges are
sometimes faced by pathologists in the distinction from
benign hepatocellular mass lesions. This is particularly
true of hepatocellular adenoma, which can sometimes be
extremely difcult to distinguish from well differentiated
1726 Arch Pathol Lab MedVol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass LesionsWang et al
Figure 2. Examples of mixed glypican-3 immunostaining patterns observed in the same hepatocellular carcinoma (A) and focal immunoreactivity
(B) (original magnications 400).
Figure 3. Examples of glypican-3 expression in hepatocellular carcinomas arising in cirrhotic (A) and noncirrhotic (B) livers (original magni-
cations 40 [A] and 100 [B]).
Table 2. Correlation of Glypican-3 Expression in
Hepatocellular Carcinoma With Tumor Differentiation
Tumor
Differentiation
No. (%) of cases
Negative Focal Diffuse Total
Well 17 (33.3) 15 (29.4) 19 (37.3) 51
Moderate 9 (20.0) 6 (13.3) 30 (66.7) 45
Poor 1 (6.7) 2 (13.3) 12 (80.0) 15
Total 27 (24.3) 23 (20.7) 61 (55.0) 111
HCC even on resection specimens.
14
It is unfortunate that
the currently available immunomarkers, such as -fetopro-
tein, polyclonal carcinoembryonic antigen, and CD34,
have signicant diagnostic limitations.
14,15
In this study, we immunohistochemically examined a
large number of hepatocellular mass lesions to further
evaluate the diagnostic value of GPC3. Our ndings ex-
tended the observations by others
811,16,17,2022
and con-
rmed that GPC3 is a useful immunomarker to help sep-
arate HCC from hepatocellular adenoma and other benign
hepatocellular mass lesions in a large cohort. Specically,
we detected GPC3 expression in 75.7% (84/111) of HCCs
included in our study, a frequency comparable to those
reported in previous studies. For example, in the study by
Yamauchi et al,
16
positive GPC3 staining was observed in
47 (83.9%) of 56 HCCs. Interestingly, all positive cases in
that study showed diffuse staining, which differs from the
observations by us and others that positive cases frequent-
ly exhibit focal immunoreactivity.
8,20,22
This may have a
considerable effect when using GPC3 immunostaining on
needle core biopsies because a much lower detection rate
should be expected because of sampling issues.
23
It is in-
teresting to note that heterogeneous GPC3 immunoreac-
tivity and different staining patterns seen within the same
tumor did not correlate with any morphologic character-
istics.
Using a 1-mm tissue microarray, Wang et al
17
detected
GPC3 expression in 38 (70.4%) of 54 HCCs. These authors
also reported that GPC3 immunoreactivity was more fre-
quently detected in HCCs with cirrhosis (19/21; 90%)
than those without (18/28; 64%). However, this is not the
case in our study where 51 (76.1%) of 67 HCCs with cir-
rhosis and 33 (75.0%) of 44 HCCs without cirrhosis were
found to express GPC3. Wang et al
17
reported a P value
of .01 for the difference in their study, but recalculation
Arch Pathol Lab MedVol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass LesionsWang et al 1727
Figure 4. An example of glypican-3 expression in nonneoplastic cir-
rhotic nodules (original magnication 200).
of their data using the
2
test with the Yates continuity
correction, as we did to our data, resulted in a P value of
.08.
Our study also demonstrated that GPC3 expression in
HCC did not correlate with the size or stage of the tumors
or with the underlying etiologies. These ndings are in
accordance with previous observations.
16,20,21
It is some-
what unclear, however, whether GPC3 expression in HCC
correlates with tumor differentiation. As observed by Ya-
mauchi et al
16
and Libbrecht et al,
20
there appears to be a
trend that well-differentiated HCCs express GPC3 less fre-
quently than moderately and poorly differentiated HCCs,
but the difference is not statistically signicant. Neverthe-
less, in the study by Di Tommaso et al,
21
the authors re-
ported that the number of GPC3-immunoreactive cells sta-
tistically increased with HCC dedifferentiation.
There have been only 3 prior studies, to our knowledge,
that have examined GPC3 expression in a total of 28 he-
patocellular adenomas.
8,16,17
In line with the observations
in those studies, we found completely negative GPC3
staining in all 48 hepatocellular adenomas included in our
study. It should be emphasized, however, that well-differ-
entiated HCC, which poses the most diagnostic difculties
in the distinction from hepatocellular adenoma, frequently
fails to express GPC3. In our series, one third of well-
differentiated HCCs were completely negative for GPC3
expression, and another third showed only focal immu-
noreactivity. Therefore, GPC3 immunostaining is helpful
only when it is positive, and negative staining should not
be viewed as evidence to exclude the possibility of HCC.
This is particularly true of needle core biopsies.
23
Inter-
estingly, Wang et al
17
detected GPC3 expression in 3 (60%)
of 5 atypical hepatocellular adenomas, suggesting that
GPC3 could be helpful in identifying cases with malig-
nant progression. However, malignant transformation ap-
pears to be a rare event because none of our adenoma
cases showed any detectable GPC3 immunostaining.
Another interesting observation in our study is the de-
tection of focal GPC3 expression in cirrhotic nodules in a
small subset of HCC cases, but not in cirrhotic livers or
even large regenerative nodules in cases without HCC.
This raises the possibility that GPC3 may function as an
early biomarker in hepatocarcinogenesis. In this regard,
several studies have attempted to examine GPC3 expres-
sion in dysplastic nodules, a poorly dened premalignant
nodular lesion arising in cirrhotic livers.
24
Overall, to our
knowledge, a total of 160 dysplastic nodules (69 low
grade, 77 high grade, 14 unspecied) have been examined
by immunohistochemistry, and only 17 (10.6%) of the nod-
ules showed positive staining, usually focal.
8,16,17,2022
Al-
though high-grade dysplastic nodules tended to express
GPC3 more frequently (13/77; 16.9%) than low-grade dys-
plastic nodules (4/69; 5.8%), no statistically signicant dif-
ference was detected (P .07). It should be pointed out
that the results reported by different investigators varied
signicantly. For example, Yamauchi et al
16
reported GPC3
expression in 2 (25%) of 8 low-grade and 6 (75%) of 8
high-grade dysplastic nodules. On the other hand, Lib-
brecht et al
20
showed no GPC3 immunostaining in 16 low-
grade dysplastic nodules and focal staining in only 2 (6%)
of 33 high-grade dysplastic nodules. One explanation for
the variable results in different studies may be the dif-
culty and wide interobserver variability in the diagnosis
of dysplastic nodules.
25
In our study, we intentionally ex-
cluded dysplastic nodules because we believed that the
results of immunochemical studies based on inaccurate or
disputable diagnoses would not be reliable or clinically
useful.
25,26
Nevertheless, it is interesting to note that the
GPC3-positive cirrhotic nodules in HCC cases in our se-
ries did not appear to exhibit dysplastic features and were
morphologically indistinguishable from GPC3-negative
nodules. Although the underlying mechanism(s) for selec-
tive GPC3 expression in cirrhotic nodules remains elusive,
it is our speculation that the GPC3-expressing hepatocytes
in cirrhotic nodules may have already initiated the neo-
plastic process at the molecular level. It is possible, there-
fore, that reactivation of GPC3 expression may represent
an early molecular event in hepatocarcinogenesis, which
precedes actual morphologic transformation.
In summary, our data demonstrate that GPC3 is a reli-
able diagnostic immunomarker, when positive, to distin-
guish HCC from benign hepatocellular mass lesions, in-
cluding hepatocellular adenoma. Our ndings emphasize
that GPC3 immunoreactivity can be focal and heteroge-
neous, and thus negative immunostaining should not ex-
clude the diagnosis of HCC. Our study also shows focal
GPC3 expression in a small subset of cirrhotic nodules
associated with HCCs, which should be taken into consid-
eration when interpreting challenging needle biopsies.
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Archives of Pathology & Laboratory Medicine and Archives of Ophthalmology will publish a joint
theme issue on ophthalmic pathology in August 2009. Articles on diagnostic procedures, path-
ologic mechanistic pathways, and translational research in retinoblastoma, melanoma, lympho-
ma, orbital, and adnexal tumors in ophthalmic pathology will have the best chance for consid-
eration in this theme issue. Manuscripts must be submitted no later than February 1, 2009 for
consideration in the joint theme issue. All submissions will undergo our usual peer review process.
Important: When submitting a manuscript for this theme issue, be certain to mention this in
the cover letter. In addition, click on Ophthalmic Pathology in the Special Section area of the
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