Arch Pathol Lab Med—Vol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass Lesions—Wang et al 1723

Original Articles

Glypican-3 as a Useful Diagnostic Marker ThatDistinguishes Hepatocellular Carcinoma From Benign

Hepatocellular Mass LesionsHanlin 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 masslesions, particularly hepatocellular adenoma, can some-times be challenging. The currently available ancillary toolsare suboptimal in terms of sensitivity and specificity.

Objective.—To further characterize the diagnostic valueof glypican-3 (GPC3), a cell surface proteoglycan that hasrecently been shown to be overexpressed in HCC, in thedistinction between HCC and benign hepatocellular masslesions.

Design.—A total of 221 surgically resected liver speci-mens were subjected to immunohistochemical staining us-ing a monoclonal antibody specific for GPC3. These in-cluded 111 HCCs, 48 hepatocellular adenomas, 30 focalnodular hyperplasias, and 32 large regenerative nodules inthe background of cirrhosis.

Results.—Cytoplasmic, membranous, and canalicularstaining for GPC3 was detected in 84 (75.7%) of the 111HCCs, among which, 61 (72.6%) of the 84 cases exhibiteddiffuse immunoreactivity. In contrast, none of the 110 cas-

es of hepatocellular adenoma, focal nodular hyperplasia,and large regenerative nodule showed detectable GPC3staining. Focal GPC3 immunoreactivity was detected incirrhotic nodules in 11 (16.4%) of 67 HCC cases with acirrhotic background, but no background staining was ob-served in the remaining 44 HCCs without cirrhosis. GPC3expression in HCCs did not correlate with the size, differ-entiation, or stage of the tumors; the presence or absenceof cirrhotic background; or the underlying etiologies.

Conclusions.—GPC3 is a specific immunomarker forHCC 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 canalso be focal, and thus, the lack of GPC3 staining does notexclude the diagnosis of HCC.

(Arch Pathol Lab Med. 2008;132:1723–1728)

Glypican-3 (GPC3), a member of the heparan sulfateproteoglycan family, is an oncofetal protein that is

expressed in the embryo and involved in morphogenesisand growth control during development.1,2 Its expressionis silenced in adult tissues, and loss-of-function mutationsare responsible for Simpson-Golabi-Behmel syndrome, arare X-linked prenatal and postnatal overgrowth withmultiple 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 ofPathology and Immunology, Washington University School of Medi-cine, St Louis, Mo (Dr Anatelli); the Department of Pathology, WeillMedical College of Cornell University, The Methodist Hospital, Hous-ton, Tex (Dr Zhai); and the Department of Pathology, NorthwesternUniversity Feinberg School of Medicine, Chicago, Ill (Drs Adley,Chuang, and Yang).

The authors have no relevant financial interest in the products orcompanies described in this article.

Presented in part at the 96th Annual Meeting of the United Statesand Canadian International Academy of Pathology, San Diego, Calif,March 24–30, 2007.

Reprints: Hanlin L. Wang, MD, PhD, Department of Pathology andLaboratory 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 thatGPC3 induces apoptosis in certain cell lines via the an-choring of the protein to the cell membrane,5 indicatingthat GPC3 may function as an inhibitor of cell proliferationand a tumor suppressor in a cell line–specific manner.

Glypican-3 as a potential tumor marker for hepatocel-lular carcinoma (HCC) was first suggested in 1997 by Hsuet al,6 who reported that GPC3 mRNA was preferentiallyexpressed in HCCs. In their study of primary and/or re-current HCCs from 154 patients, GPC3 (named MXR7 intheir study) mRNA was detected in 143 (74.8%) of the 191tumors, in contrast to only 3 cirrhotic and 2 (3.2%) of thenoncirrhotic adult livers that showed low levels of GPC3mRNA expression. In addition, GPC3 mRNA was not de-tected in 3 hepatocellular adenomas included in theirstudy. These observations were subsequently confirmedby Zhu et al7 in a similar study.

In 2003, several studies showed increased GPC3 ex-pression at the protein level in HCC, when compared withhealthy livers and benign hepatic lesions.8–11 In the studyby Capurro et al,8 GPC3 protein expression was demon-strated by immunohistochemistry in 21 (72%) of 29 HCCcases but was not detected in healthy livers or variousbenign liver lesions, including 22 cirrhotic livers, 7 hepa-

1724 Arch Pathol Lab Med—Vol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass Lesions—Wang et al

tocellular adenomas, and 4 focal nodular hyperplasias. Inaddition, GPC3 can be detected in the serum as a secretedprotein in a subset of patients with HCC but is undetect-able in healthy individuals or patients with hepatitis orcirrhosis.8,10,12,13 Interestingly, elevated serum GPC3 levelsin 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 wouldimprove overall sensitivity in HCC detection.

One of the challenges in histopathologic diagnosis ofhepatocellular mass lesions is to distinguish HCC (partic-ularly well differentiated) from hepatocellular adenoma,and the available immunomarkers in clinical practice, suchas �-fetoprotein, polyclonal carcinoembryonic antigen,and CD34, have significant diagnostic limitations.14,15 Inthis regard, GPC3 has shown promise as a useful diag-nostic immunomarker as demonstrated in 3 studies inwhich a total of 28 hepatocellular adenomas were immu-nohistochemically examined.8,16,17 In the current study, alarge 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 thisstudy. These included 111 HCCs, 48 hepatocellular adenomas, 30focal nodular hyperplasias, and 32 large regenerative nodulesarising in cirrhotic livers. These cases were retrieved from sur-gical pathology archives of Washington University Barnes–JewishHospital 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 withhematoxylin-eosin were reviewed to confirm the diagnoses andto determine the underlying etiologies, when applicable. Hepa-tocellular carcinoma cases, in which the patients had undergonepreoperative chemoembolization with tumor necrosis, were ex-cluded from this study.

Immunohistochemical staining for GPC3 was performed fol-lowing the protocol described previously.18 Briefly, 4-�m sectionsfrom formalin-fixed, paraffin-embedded tissue blocks were de-paraffinized, rehydrated, and treated with 3% hydrogen peroxidefor 15 minutes to inhibit endogenous peroxidase. Following mi-crowave heat-induced epitope retrieval in 0.1M citrate buffer atpH 6.0 for 20 minutes, the slides were incubated with a mousemonoclonal antibody specific for GPC3 (clone 1G12) obtainedfrom BioMosaics (Burlington, Vt), with a dilution of 1:200 for 1hour at room temperature. After incubation with a rabbit, anti-mouse secondary antibody, a reaction was performed using theEnVision� detection system that contains biotin-free horseradishperoxidase–labeled polymers obtained from Dako Corporation(Carpinteria, Calif). The staining was visualized using 3,3�-dia-minobenzidine substrate–chromogen solution and counterstainedwith hematoxylin.

Immunohistochemically stained slides were independentlyevaluated by 3 observers (H.L.W., F.A., and X.J.Y.). Cases withsignificantly discrepant interpretation were resolved by rereviewtogether by 2 of the observers (H.L.W. and F.A.). A case wasconsidered negative if �5% of the cells of interest exhibited im-munoreactivity. Positive stains were further stratified as focal (5%to 50% of the cells stained) and diffuse (�50% of the cellsstained). 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 Pvalue of �.05 was considered statistically significant.

RESULTS

Clinicopathologic Features of Hepatocellular Lesions

The ages of patients with HCC ranged from 26 to 82years (mean, 60.1 years; median: 62 years). Of the 111 pa-tients, 70 were men, and 41 were women, for a male tofemale 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 caseswere 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%) ofthe tumors were detected in a cirrhotic background,whereas 44 (39.6%) arose in noncirrhotic livers. The un-derlying etiologies associated with HCC developmentcould not be determined in 33 cases (29.7%), amongwhich, 78.8% (26/33) of the HCCs arose in noncirrhoticlivers. In the remaining cases, hepatitis C was documentedin 42 (37.8%) cases, hepatitis B in 12 cases (10.8%), hepa-titis B and C coinfection in 2 cases (1.8%), hepatitis B andC 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 deficiency in1 case (0.9%).

The ages of patients with hepatocellular adenomaranged from 10 to 83 years (mean, 36.8 years; median, 33years). 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 surgicalresection, 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 hyperplasiaranged from 9 to 78 years (mean, 37.2 years; median, 38years). 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 ofusing oral contraceptives. At the time of surgical resection,the lesions ranged in size from 0.3 to 14.0 cm (mean, 4.3cm; median, 4.3 cm).

The ages of patients with large, regenerative nodules ina 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 maleto 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 deficiency(2/32; 6.3%), primary sclerosing cholangitis (1/32; 3.1%),and cryptogenic origin (9/32; 28.1%).

Immunohistochemical Findings of GPC3 Expression inHepatocellular Lesions

As shown in Table 1, positive immunostaining for GPC3was observed in 84 (75.7%) of the 111 HCC cases, amongwhich, 61 (72.6%) cases showed diffuse immunoreactivity.In marked contrast, none of the 110 benign hepatocellularmass 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 Med—Vol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass Lesions—Wang et al 1725

Table 1. Summary of Immunohistochemical Findingsof 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 (5–50) 23 (20.7) 0 0 0Diffuse (�50) 61 (55.0) 0 0 0

* HCC indicates hepatocellular carcinoma; HCA, hepatocellular ad-enoma; FNH, focal nodular hyperplasia; and LRN, large regenerativenodule.

Figure 1. Examples of different glypican-3 immunostaining patternsobserved in hepatocellular carcinomas: (A) predominantly membra-nous, (B) predominantly canalicular, and (C) predominantly cytoplas-mic (original magnifications 400).

patterns were observed: predominantly membranous (Fig-ure 1, A), predominantly canalicular (Figure 1, B), andpredominantly cytoplasmic (Figure 1, C). These differentstaining patterns did not appear to correlate with the dif-ferentiation status or the growth pattern of the tumors. Infact, more than one third of the tumors exhibited a mixedstaining pattern within the same tumors, although the tu-mor cells with different staining patterns showed similaror identical histologic features (Figure 2, A). Similarly, inthose tumors that were focally positive for GPC3 expres-sion, the tumor cells showing negative immunoreactivitydid not appear to be dissimilar morphologically to thosewith 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 thanwell-differentiated HCCs (93.3% vs 66.7%), the differencedid not reach a statistical significance (P .05). In addi-tion, HCCs arising in cirrhotic livers expressed GPC3 at asimilar 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 ofthe 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 HCCsexhibited GPC3 immunoreactivity. There was only 1 caseof sarcomatoid HCC included in our study, which showedfocal GPC3 expression. There was also only 1 case of fi-brolamellar HCC, which showed a diffuse staining pat-tern.

Among 67 HCCs with a cirrhotic background, focalGPC3 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 weremorphologically indistinguishable from the hepatocytesthat did not stain for GPC3, either in the same nodules orin different nodules (Figure 4). The HCCs in these 11 caseswere all positive for GPC3 expression, 7 (63.6%) diffuseand 4 (36.4%) focal, typically with a stronger staining in-tensity relative to that detected in cirrhotic nodules. Nobackground staining was observed in nonneoplastic he-patocytes in any of the 44 HCC cases without cirrhosis.

COMMENTHCC is the fifth most common cancer and the third

most common cause of cancer-related deaths worldwide.19

Accurate diagnosis is critically important to appropriateclinical management of the patients and assessment of the

prognosis. Although histopathologic diagnosis of HCCmay be straightforward in many cases, challenges aresometimes faced by pathologists in the distinction frombenign hepatocellular mass lesions. This is particularlytrue of hepatocellular adenoma, which can sometimes beextremely difficult to distinguish from well differentiated

1726 Arch Pathol Lab Med—Vol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass Lesions—Wang et al

Figure 2. Examples of mixed glypican-3 immunostaining patterns observed in the same hepatocellular carcinoma (A) and focal immunoreactivity(B) (original magnifications 400).

Figure 3. Examples of glypican-3 expression in hepatocellular carcinomas arising in cirrhotic (A) and noncirrhotic (B) livers (original magnifi-cations 40 [A] and 100 [B]).

Table 2. Correlation of Glypican-3 Expression inHepatocellular Carcinoma With Tumor Differentiation

TumorDifferentiation

No. (%) of cases

Negative Focal Diffuse Total

Well 17 (33.3) 15 (29.4) 19 (37.3) 51Moderate 9 (20.0) 6 (13.3) 30 (66.7) 45Poor 1 (6.7) 2 (13.3) 12 (80.0) 15Total 27 (24.3) 23 (20.7) 61 (55.0) 111

HCC even on resection specimens.14 It is unfortunate thatthe currently available immunomarkers, such as �-fetopro-tein, polyclonal carcinoembryonic antigen, and CD34,have significant diagnostic limitations.14,15

In this study, we immunohistochemically examined alarge number of hepatocellular mass lesions to furtherevaluate the diagnostic value of GPC3. Our findings ex-tended the observations by others8–11,16,17,20–22 and con-firmed that GPC3 is a useful immunomarker to help sep-arate HCC from hepatocellular adenoma and other benignhepatocellular mass lesions in a large cohort. Specifically,we detected GPC3 expression in 75.7% (84/111) of HCCs

included in our study, a frequency comparable to thosereported in previous studies. For example, in the study byYamauchi et al,16 positive GPC3 staining was observed in47 (83.9%) of 56 HCCs. Interestingly, all positive cases inthat study showed diffuse staining, which differs from theobservations by us and others that positive cases frequent-ly exhibit focal immunoreactivity.8,20,22 This may have aconsiderable effect when using GPC3 immunostaining onneedle core biopsies because a much lower detection rateshould 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 sametumor did not correlate with any morphologic character-istics.

Using a 1-mm tissue microarray, Wang et al17 detectedGPC3 expression in 38 (70.4%) of 54 HCCs. These authorsalso 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 thecase in our study where 51 (76.1%) of 67 HCCs with cir-rhosis and 33 (75.0%) of 44 HCCs without cirrhosis werefound to express GPC3. Wang et al17 reported a P valueof �.01 for the difference in their study, but recalculation

Arch Pathol Lab Med—Vol 132, November 2008 Diagnostic Value of GPC3 in Liver Mass Lesions—Wang et al 1727

Figure 4. An example of glypican-3 expression in nonneoplastic cir-rhotic nodules (original magnification 200).

of their data using the �2 test with the Yates continuitycorrection, as we did to our data, resulted in a P value of.08.

Our study also demonstrated that GPC3 expression inHCC did not correlate with the size or stage of the tumorsor with the underlying etiologies. These findings are inaccordance with previous observations.16,20,21 It is some-what unclear, however, whether GPC3 expression in HCCcorrelates with tumor differentiation. As observed by Ya-mauchi et al16 and Libbrecht et al,20 there appears to be atrend that well-differentiated HCCs express GPC3 less fre-quently than moderately and poorly differentiated HCCs,but the difference is not statistically significant. 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 observationsin those studies, we found completely negative GPC3staining in all 48 hepatocellular adenomas included in ourstudy. It should be emphasized, however, that well-differ-entiated HCC, which poses the most diagnostic difficultiesin the distinction from hepatocellular adenoma, frequentlyfails to express GPC3. In our series, one third of well-differentiated HCCs were completely negative for GPC3expression, and another third showed only focal immu-noreactivity. Therefore, GPC3 immunostaining is helpfulonly when it is positive, and negative staining should notbe viewed as evidence to exclude the possibility of HCC.This is particularly true of needle core biopsies.23 Inter-estingly, Wang et al17 detected GPC3 expression in 3 (60%)of 5 atypical hepatocellular adenomas, suggesting thatGPC3 could be helpful in identifying cases with malig-nant progression. However, malignant transformation ap-pears to be a rare event because none of our adenomacases showed any detectable GPC3 immunostaining.

Another interesting observation in our study is the de-tection of focal GPC3 expression in cirrhotic nodules in asmall subset of HCC cases, but not in cirrhotic livers oreven large regenerative nodules in cases without HCC.This raises the possibility that GPC3 may function as anearly biomarker in hepatocarcinogenesis. In this regard,

several studies have attempted to examine GPC3 expres-sion in dysplastic nodules, a poorly defined premalignantnodular lesion arising in cirrhotic livers.24 Overall, to ourknowledge, a total of 160 dysplastic nodules (69 lowgrade, 77 high grade, 14 unspecified) have been examinedby immunohistochemistry, and only 17 (10.6%) of the nod-ules showed positive staining, usually focal.8,16,17,20–22 Al-though high-grade dysplastic nodules tended to expressGPC3 more frequently (13/77; 16.9%) than low-grade dys-plastic nodules (4/69; 5.8%), no statistically significant dif-ference was detected (P .07). It should be pointed outthat the results reported by different investigators variedsignificantly. For example, Yamauchi et al16 reported GPC3expression in 2 (25%) of 8 low-grade and 6 (75%) of 8high-grade dysplastic nodules. On the other hand, Lib-brecht et al20 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 forthe variable results in different studies may be the diffi-culty and wide interobserver variability in the diagnosisof dysplastic nodules.25 In our study, we intentionally ex-cluded dysplastic nodules because we believed that theresults of immunochemical studies based on inaccurate ordisputable diagnoses would not be reliable or clinicallyuseful.25,26 Nevertheless, it is interesting to note that theGPC3-positive cirrhotic nodules in HCC cases in our se-ries did not appear to exhibit dysplastic features and weremorphologically indistinguishable from GPC3-negativenodules. Although the underlying mechanism(s) for selec-tive GPC3 expression in cirrhotic nodules remains elusive,it is our speculation that the GPC3-expressing hepatocytesin 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 representan early molecular event in hepatocarcinogenesis, whichprecedes 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 findings emphasizethat GPC3 immunoreactivity can be focal and heteroge-neous, and thus negative immunostaining should not ex-clude the diagnosis of HCC. Our study also shows focalGPC3 expression in a small subset of cirrhotic nodulesassociated with HCCs, which should be taken into consid-eration when interpreting challenging needle biopsies.

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