Glypican-3 is a useful diagnostic marker for a component of hepatocellular carcinoma in human liver cancer

Primary liver cancers are classified into three types based on their morphology and cytogenetic characteristics hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular and cholangiocarcinoma (CHC). It is often difficult to distinguish these liver tumors. Glypican-3 (GPC3) is serological and histochemical marker of hepatocellular carcinoma. In order to separate these three types of liver cancers, we analyzed the GPC3 expression in 85 liver resection specimens, including 46 HCCs, 28 ICCs and 11 CHCs. GPC3 immunohistochemical staining was used to distinguish HCC from ICC by comparing with the conventional biomarker, ·-fetoprotein (AFP). The immunostaining of GPC3 was identified in 78.3% (36/46) of HCCs, 60% (9/15) of well differentiated, 88.9% (16/18) of moderately differentiated and 84.6% (11/13) of poorly differentiated HCCs. It was negative in the ICCs. We confirmed that GPC3 expression is specific to HCC component (8/11, 72.7%) but few samples also showed weakly in ICC component (2/11, 18.2%) of CHC sections among 11 cases compared with HCC biomarkers including AFP and hepatocyto paraffin 1 (HepPar1), and ICC biomarkers cytokeratin (CK) 7 and CK19. Three cases in which the macroscopic features resembled ICC did not express GPC3 even in the pathological HCC component. Most (10/11, 91%) of the pathological cholangiocarcinoma components in CHC showed positive staining for CK7 and CK19. The results of this study suggest that GPC3 is a biomarker that is sensitive and specific to HCC component of CHC, and CK7 and CK19 are markers for pathological cholangiocarcinoma component of CHC.


Introduction
Liver cancer is one of the common malignancies that are rapidly increasing throughout the world.Primary liver cancers are classified into three types based on their morphology and cytogenetic characteristics, hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular and cholangiocarcinoma (CHC).HCC is hepatocyto-origin, and ICC is from the epithelium of the intrahepatic bile duct.CHC is a rare type of liver cancer with features of both hepatocellular and biliary differentiation (1)(2)(3).The pathological structure of CHC is composed of hepatocellular element showing bile production, an intercellular bile canaliculi or trabecular growth pattern and cholangiocellular component showing mucin production or gland formation.
Because of their rapid growth rate and the lack of accurate ways of diagnosis in the early stages, the prognosis and the survival rate for liver cancer patients remain poor.Currently, ultrasound sonography (US), computed tomography (CT), magnetic resonance imaging (MRI), and histopathological examination for tumor biopsy are used for diagnosis.However, distinguishing the three different primary liver tumors is often a challenging task in diagnosis, for which immunohistochemical analysis for specific antigens is a helpful tool: •-fetoprotein (AFP) and hepatocyto paraffin 1 (HepPar1) for HCC (4)(5)(6)(7)(8) and cytokeratin (CK) 7 and CK19 for ICC (9)(10)(11).
Glypican-3 (GPC3) was discovered as a potential serological and histochemical marker whose expression is specific for HCC (12)(13)(14)(15)(16). GPC3 belongs to glypican family that is a group of heparan sulfate proteoglycans linked to the outer surface of cell membrane through a glycosylphosphatidylinositol anchor (17).In mammals, six members of GPCs have been reported, GPC1 to GPC6.GPCs are released from the cell surface by a lipase called Notum to regulate the signaling of Wnts, Hedgehogs, fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs) (18)(19)(20)(21)(22)(23)(24)(25).Depending on the cellular context, their function can be stimulatory or inhibitory activity, or signaling.The expression of GPC3 is detected in placenta and fetal liver, but not in other normal organs.During hepatic carcinogenesis, GPC3 have been reported to reappear in HCC and to be released into serum (12,13,15,26).Its expression is also detected in melanoma (27)(28)(29).The functions of GPC3 in cancer cells are still unclear.
In this study, we examined whether immunohistochemical analysis for GPC3 can be used to distinguish HCC from ICC, if so, how effectively GPC3 can be detected, compared to other biomarkers that are conventionally used.We demonstrate that distinguishing HCC from ICC by detecting the expression of GPC3 enables more accurate diagnosis.

Materials and methods
Case selection.We selected 85 cases of liver tumors from the surgical pathology files from 1992 to 2006 of National Cancer Center Hospital East, Kashiwa, Chiba, Japan.The cases included 46 primary HCCs, 28 ICCs, and 11 CHCs that underwent hepatectomy.All identifiers were eliminated to protect patients' identities.Size of the tumor and any clinicopathologic factors (age, sex and grade of tumor) were matched between HCC and ICC.The 46 cases of HCCs occurred in 33 men and 13 women with a mean of age at 65.3 years (range, 44-80 years).HCC was subclassified into well (n=15), moderately (n=18), and poorly (n=13) differentiated types according to the World Health Organization classification criteria.The 28 cases of ICC consisted of 18 men and 10 women.Their mean age was 65.7 years (range, 51-82 years).All 28 resected cases of ICC were confirmed by hematoxylin-eosin (H.E.) staining.
The 11 cases of CHC included 7 men and 4 women with a mean age of 62.5 years (range, 47-76 years).All CHCs were pathologically confirmed after surgery.Tissue samples.Liver tissue sections were retrieved from the files of the Department of Pathology in our institution.All liver specimens were prepared from surgically resected tumors and adjacent parenchyma.They were fixed in 10% formalin and paraffinized for routine histological examination.
Immunohistochemical staining procedure.Six-micrometerthick sections were made from the paraffin-embedded blocks.Subsequently the sections were deparaffinized in xylene and rehydrated through ethanol to water.Endogenous peroxidase activity was blocked using 3% H 2 O 2 in methanol for 20 min.For antigen retrieval, Sections were heated in 10 mM citrate buffer (pH 6.0) with microwave for 15 min in a water bath at 95˚C.Only for CK7 immunostaining, sections were digested by Proteinase K (DakoCytomation, Carpenteria, CA) for 5 min at room temprature.Slides were then allowed to cool down.The prediluted primary antibodies, monoclonal anti-GPC3 (dilution 1:300, 1G12; Biomosaics, Inc., Burlington, VT), anti-AFP (dilution 1:400, Dako-Cytomation), anti-HepPar1 (dilution 1:100, DakoCytomation), anti-CK7 (dilution 1:100, DakoCytomation), and CK19 (dilution 1:200, DakoCytomation) were added to cover each slide, and the slides were incubated for 2 h at room temperature.Slides were washed 3 times in phosphatebuffered saline (PBS)/Tween for 5 min each.Mouse Envision Polymer (DakoCytomation) was used as a secondary antibody for 30 min at room temperature followed by washes in PBS/Tween 3 times for 5 min each.Diaminobenzidine chromagen (DakoCytomation) was added to each slide and incubated for 2 min.Slides were washed in distilled water, counterstained with hematoxylin and dehydrated in xylene.To analyze GPC3 expression, the immunohistochemical results were classified according to the number of positive cells as follows: -, negative (<10%); ±, weakly positive (10-30%); + positive (>30%).To validate the data in GPC3 as a marker for HCC, parallel staining for AFP of 46 cases were further analyzed.For 11 CHC cases, AFP, HepPar1, CK7 and CK19 were stained and compared with GPC3 staining pattern.
The slides were examined independently by 3 observers (Shirakawa H, Kuronuma T and Nakatsura T) and then collectively by 2 more pathologists (Hasebe T and Nakano M).
Statistical analysis.Differences in proportion were tested by the ¯2 test.Differences in the means of each subgroup were tested using the Student's t-test.P-value of <0.05 was considered statistically significant.

GPC3 was present in 80% of HCC and negative in ICC.
In order to examine the levels and pattern of GPC3 expression, 46 cases of HCC and 28 cases of ICC were immunohistochemically analyzed.GPC3 was detected in 36 cases (78%) of HCC (Fig. 1a), and no expression of GPC3 was found in any of the ICC patients (Fig. 1b).The GPC3 staining was diffused throughout (Fig. 1c) or localized in a granular pattern in the cytoplasm (Fig. 1d).In other cases, GPC3 was observed at the plasma membrane (Fig. 1e).Previously GPC3 is shown to bind to the cell membrane ( 16), however, those cases with membranous GPC3 had staining in the cytoplasm as well, but there was no case of GPC3 located only at the plasma membrane.When sensitivity of GPC3 was evaluated, 36 cases (78%) were positive for GPC3 when only 16 cases (35%; P<0.0001) were stained for AFP in HCC suggesting that GPC3 is more sensitive than AFP.Thus, GPC3 was confirmed to be specific and sensitive to HCC compared to AFP.

GPC3 expression increased in moderately and poorly differentiated HCC.
In terms of GPC3 expression and tumor differentiation level, GPC3 was expressed in 9 (60%) of 15 well differentiated, 16 (89%) of 18 moderately differentiated and in 11 (85%) of 13 poorly differentiated HCC (Table I).AFP was expressed in 3 (20%) of 15 well differentiated, 6 (33%) of 18 moderately differentiated and in 7 (54%) of 13 poorly differentiated HCC (data not shown).The expression level of GPC3 was lower in well differentiated HCC than in the other HCC grades, though the difference was not statistically significant (well-vs.moderately differentiated: P=0.054, well-vs.poorly differentiated: P=0.150).Thus, GPC3 expression is also a good indicator for malignancy levels.

GPC3 expression was observed specifically in pathological HCC component in CHC.
There are discrepancies between preoperative diagnosis and pathological findings for CHC patients.Diagnostic results and the expression of tumor markers of 11 CHC patients are summarized in Table II.Initial diagnosis was carried out by H.E. staining.Among these 11 patients, 7 patients (63.6%) were diagnosed as HCC and 3 (27.3%)were ICC.Only 1 patient (9%) of the 11 CHC was correctly diagnosed as CHC.To seek the possibility to use GPC3 immunostaining to detect HCC component (cp) in CHC, combination of antibodies against GPC3, AFP, HepPar1, CK7 and CK17 were used.In addition to AFP, HepPar1 is frequently used as marker for HCC (4-8) and CK 7 and CK19 for ICC (9)(10)(11).
Case 6 showed pale and lobulated phenotype in the cut surface macroscopically (Fig. 2b-i), and pathological diagnosis was also confirmed by H.E. staining (Fig. 2b-ii and -iii).This was so-called 'transitional' type tumor (30).A 'transitional' type tumor has an area of HCC that appears to transform into CC (31).GPC3 was stained in pathological HCC cp (Fig. 2b-iv) where AFP was negative (Fig. 2b-v).The HCC region was surrounded by pathological CC cp with the staining for CK7 (Fig. 2b-vii).HepPar1 and CK19 were detected in the same region with CC cp (Fig. 2b-vi and -viii).HepPar1 stained the CC cp as in case 2. The immunoreactivity of CK19 was not consistent with that of CK7.
Case 8 was diagnosed as HCC similarly to cases 2 and 6, but mixed tumor masses with white and gray in the cut surface were observed (Fig. 2c-i and c-ii).Both GPC3 and AFP were positive in HCC cp (Fig. 2c-iv and -v).HepPar1 was stained in CC cp (Fig. 2c-vi).CK7 and CK19 were positive in CC cp (Fig. 2c-vii and -viii), especially CK19 was more specific for CC cp than CK7.These three cases (cases 2, 6 and 8) indicated that detecting GPC3 can compensate for AFP and enhance the ability to identify the presence of HCC cp in CHC.
Cases 9, 10 and 11 were negative for GPC3 expression in several tumors.Macroscopically, they had the features of ICC with irregular shaped, white solid tumor masses.As an example, case 10 is shown in Fig. 2d.Althogh case 10 was diagnosed as HCC preoperativerly, it showed macroscopic freatures of ICC with the presence of abundant fibrous stroma and indistinct tumor margin (Fig. 2d-i).This case was later diagnosed as CHC based on the pathological examination (Fig. 2d-ii and d-iii).GPC3, AFP and HepPar1 were not detected in either HCC cp or CC cp (Fig. 2d-iv, -v, and -vi).CK7 was stained diffusely in the tumor (Fig. 2dvii), and CK19 expression was more specific in CC cp than CK7 (Fig. 2d-viii).These 3 cases showed positive staining Table II.Correlation of immunostaining varieties and pathological components of CHC.

Discussion
The diagnosis for HCC, ICC and CHC has been routinely performed by histopathological examination.Additionally, diagnosis of HCC is done by supplementary immunohistochemical analysis for AFP and HepPar1.Until now, though the sensitivity is limited, AFP has been regarded as the most useful marker for HCC (4,(32)(33)(34).HepPar1 is also widely used for HCC to distinguish between primary HCC and ICC.However, both markers are limited for the ability to discriminate different levels of malignancy in HCC because its sensitivity drops substantially in poorly differentiated HCC, and it does not discriminate between benign and malignant liver cancers (35).As these biomarkers frequently results in misdiagnosis, in this study, we showed that GPC3 is more sensitive to detect HCC compared to AFP.Due to the fact that GPC3 was downregulated in ICC (36), GPC3 may help to separate HCC from ICC.
CHC is the least common primary cancer of the liver but followed by an aggressive growth, it tends to metastasize to many organs leading to significantly poorer prognosis than HCC and ICC (31,37,38).Correct diagnosis leads to both appropriate treatment and better outcome for the patients.Nishie, et al reported that one third (nine of 27 cases) of patients with CHC were correctly diagnosed by enhanced computed tomography (39).In our study, only one of the 11 (9.1%) patients with CHC was correctly diagnosed before operation without fine needle aspiration biopsy.The difficulty to pathologically distinguish CHC from HCC and ICC comes from glandular or pseudoglandular structures in HCC and solid or trabecular patterns in CC (37,38).We believe that combination with histopathological examination with GPC3 immunostaining and radiological examination can bring an accurate diagnosis and improved clinical therapies for the patients leading to a better prognosis.
We showed that the immunostaining for GPC3 is specific for HCC patients and not detected in ICC patients.This confirmed that detecting GPC3 may improve the method to diagnose CHC.Of the 11 cases of CHC, 8 displayed GPC3 expression in restricted area of HCC cp.We demonstarated that immunohistochemical staining of GPC3 in liver tumor helps to recognize the pathological HCC cp more precisely.GPC3 expression was observed with high frequency in the HCC cp compared with AFP and HepPar1.HepPar1 was unexpectedly stained in CC cp, but this has been observed previously as well (7,40).This could be due to a transition from HCC to ICC where HepPar1 is one of the molecules that is downregulated at later stages in the process.CK7 and CK19 have been already reported as good markers of biliary epithelial differentiation (41).These were highly expressed in pathological CC cp (10/11, 91%) in CHC.The positive immunoreactivity of CK19 was more distinct than that of CK7 whose staining was weaker.Our immunohistochemical data disclosed that GPC3 can be a better marker specific for HCC leading to a better confirmation for HCC component of CHC as well as for HCC.Moreover, it provided evidence of the biologic behavior of such combined tumors, which are phenotypically and genetically leaning toward either ICC with predominant biliary differentiation or HCC with hepatocellular differentiation (42,43).
Employing multiple tumor markers may also allow the accurate diagnosis of CHC containing both hepatocellular and biliary differentiation.Concerning sensitivity and specificity, the combination of GPC3 for HCC cp and CK19 for ICC cp seems to be useful in the diagnosis of liver cancer.
For CHC, GPC3 positive/CK19 negative profile suggests HCC, GPC3 positive/CK19 positive indicates CHC, and GPC3 negative/CK19 positive essentially rules out HCC and suggests the possibility of CC or CHC.
We developed a new anti-cancer immunotherapy with GPC3 as a target (44)(45)(46)(47), and the phase I clinical trial of GPC3-derived peptide vaccination for advanced HCC is now on going.Because this new immunotherapy is not indicated for ICC, immunohistochemical staining of GPC3 is a useful method to select eligible patients.Furthermore, if CHC would be justified as a target of our immunotherapy in future, immunohistochemical analysis for GPC3 expression is indispensable for the process of patient selection.
GPC3 is expressed in the group of cells that are AFPpositive and/or CK7/19-positive in injured livers with activation of oval cell compartment; an indication for liver repair and regeneration (48).In addition, CK7, CK19 and AFP are frequently expressed in biliary epithelial cells (49,50) and in immature fetal hepatoblasts (51,52).Liver progenitor cells originate from the canal of Hering, lined by both hepatocytes and biliary ductular epithelial cells (53).It is not clear whether GPC3 is expressed in hepatic embryonic progenitor cells or cancer stem cells, but GPC3 may be a marker for hepatic progenitor/stem cells.In CHC cases of 2, 3 and 4, GPC3, CK7 and CK19 coincided in the regions of HCC and CC.Although HCC and ICC are two different kinds of primary liver malignancies arising from different cell types as hepatocytes and cholangiocytes, co-localization of GPC3 and CK7/19 suggest that the CHC is originated from progenitor or oval cell.In addition, case 6 showed an HCC lesion with GPC3 positive immunostaining surrounded by CC (Fig. 2b).This finding suggests that GPC3-positive HCC tumor cells are derived from GPC3-negative CC mass.Moreover, we predict from the fact that GPC3 is expressed in embryonic liver and downregulated after birth in normal liver but reappears in cancer is due to its regulatory role in proliferative and dedifferentiated cells, like cancer cells that acquired a progenitor-or cancer stem cell-like characteristics.
In summary, we confirmed that GPC3 is a marker sensitive and specific for HCC, but not ICC.Moreover, we revealed that GPC3 was expressed specifically in the HCC cp in the CHC.Therefore, GPC3 is a molecule that is significant not only in clinical but also biological field.It is clinically an important biomarker that can be used for accurate diagnosis leading to a better treatment and prognosis.Also, biologically, it may be an indicator for the identity and the origin of the cancer cells.

Figure 2 .
Figure 2. Macroscopic, histological and immunohistochemical features of four cases of CHC, a, case 2; b, case 6; c, case 8; d, case 10 in Table II.(a-i) Macroscopic feature in cut surface of case 2 tumor.(a-ii) The histological structure can be also devided into 2 types.HCC component showed expansive growth oppressing the cholangiocarcinoma component.(a-iii) Collision border between hepatocellular carcinoma and cholangiocarcinoma component are indicated as dots.The tumor cells within mainly hepatocellular carcinoma component showed only expression of GPC3 (a-iv) without expression of AFP (a-v).In the opposite side, the glandular area with cholangiocarcinoma component shows HepPar1 (a-vi), CK7 (a-vii) and CK19 expression (a-viii).(b-i) Case 6 shows macroscopic CHC feature in tumor cut surface that was suspected out HCC preoperatively.(b-ii) The histological cholangiocarcinoma component forming trabeculae with columnar appearance was surrounded by HCC component forming hepatoid structure.(b-iii) A dotted line is a boundary of HCC in the H.E. staining.The tumor cells within transitional region were positive for GPC3 (b-iv), CK 7 (b-vii) and CK 19 (b-viii).The difference was recognized between hepatocellular carcinoma component and cholangiocarcinoma component because GPC3 positive area encircled the CK7 area.The expressions of AFP (b-v) and HepPar1 (b-vi) were not observed.(c-i) Though case 8 was also suspected to be HCC preoperatively, the macroscopic features showed atypical HCC with mixed white and gray and indistinct tumor border.(c-ii) The cholangiocarcinoma component was obviously composed of structural gland formation.(c-iii) Collision area was distinguished histopathologically by a dotted line.The tumor cells of HCC component showed not only GPC3 (c-iv) but also AFP expression (c-v).In the glandular area of cholangiocarcinoma component, HepPar1 was expressed (c-vi), but CK7 not at all (c-vii) and CK19 shows weak positive expression (c-viii).(d-i) Case 10 shows macroscopic ICC features in tumor cut surface that was suspected as HCC preoperatively.(d-ii) The histological structure can be devided into 2 types with cholangiocarcinoma component forming trabeculae with columnar appearance and HCC component forming hepatocellular structures.(d-iii) A dotted line is a boundary of HCC in the H.E. GPC3 (d-iv), AFP (d-v) and HepPar1 (d-vi) were not stained, but CK7 (d-vii) and CK19 (d-viii) stained the cholagiocarcinoma component.