Agrin and CD34 Immunohistochemistry for the Discrimination of Benign Versus Malignant Hepatocellular...

Agrin and CD34 Immunohistochemistry for theDiscrimination of Benign Versus Malignant

Hepatocellular Lesions

Peter Tatrai, PhD,*w Aron Somoracz, MD,*w Enkhjargal Batmunkh, MD,wPeter Schirmacher, MD, DSc,z Andras Kiss, MD, PhD,w Zsuzsa Schaff, MD, DSc,w

Peter Nagy, MD, DSc,* and Ilona Kovalszky, MD, DSc*

Abstract: Agrin is a recently identified proteoglycan component

of vascular and bile duct basement membranes in the liver. The

selective deposition of agrin in hepatocellular carcinoma (HCC)

microvessels versus sinusoidal walls prompted us to investigate

the utility of agrin immunohistochemistry (IHC) in detecting

malignant hepatocellular lesions. We focused on the differential

diagnostic problems often presented by hepatocellular ade-

nomas (HCAs) and dysplastic nodules. IHC for agrin was

performed on 138 formalin-fixed, paraffin-embedded surgical

specimens from 93 patients, including cirrhotic liver tissues (25),

focal nodular hyperplasia (10), large regenerative nodules (8),

low-grade (23) and high-grade (7) dysplastic nodules, small

HCC (8), HCC (27), and HCA (30). Agrin immunostaining was

compared with that of CD34 and, in selected cases, to glypican-

3. The combination of agrin and CD34 sensitively (0.94) and

specifically (0.93) identified lesions judged previously as

malignant by histology. The majority of benign lesions were

clearly agrin-negative, whereas the strength and extent of agrin

IHC faithfully reflected dysplasia in ‘‘atypical’’ HCAs and in

high-grade dysplastic nodules. Malignant lesions were uniformly

positive. In conclusion, as agrin is highly selective for tumor

blood vessels, IHC for agrin facilitates the discrimination of

benign and malignant hepatocellular lesions. Moreover, whereas

glypican-3 in some HCCs may appear in few scattered cells only,

agrin is diffusely deposited in virtually all malignant lesions,

which may prove advantageous in the evaluation of small

specimens such as core biopsies.

Key Words: agrin, hepatocellular carcinoma, hepatocellular

adenoma, dysplastic nodule, differential diagnosis

(Am J Surg Pathol 2009;33:874–885)

D iagnosis of focal hepatocellular lesions is still acomplex issue which, despite radical advances in

medical imaging, often relies on histopathology.10 Histo-logy-based diagnosis of hepatocellular carcinoma (HCC),however, does not always lack a moment of subjectivity.No strictly objective histologic criteria have been laiddown to distinguish small HCCs (<2 cm in diameter)and high-grade dysplastic nodules (HGDNs).11 Similarly,borderline lesions between atypical hepatocellular adeno-ma (HCA) and well-differentiated HCC exist.25

Early attempts to resolve these diagnostic ambi-guities using immunohistochemistry (IHC) focused onchanges in vascular phenotype that accompany theprocess of carcinogenesis. The patterns of smooth musclealpha actin, CD31, and CD34 immunoreactions wereanalyzed to spot the differences between cirrhosis, low-grade dysplastic nodule (LGDN), HGDN, and HCC.15,18

A more recent approach concentrates on the presence orabsence of cytokeratin-7/19-positive ductular reactionaround nodular lesions: it has been suggested that thescantness or lack of ductular reaction helps to recognizeearly and overtly invasive small HCCs.16

A major advance in the immunohistochemicaldiagnosis of HCC was brought about by glypican-3, aglycosyl–phosphatidylinositol-anchored cell surface he-paran sulfate proteoglycan that is now generally acceptedas a standard IHC marker and a promising supplemen-tary serum marker of HCC.4,24 However, even glypican-3may occasionally prove inadequate for a confidentdiagnosis of HCC. Although glypican-3 IHC has beenshown to distinguish HCC from adenoma and small HCCfrom nonmalignant nodular lesions,13,23 only 63.6% ofHCCs were found glypican-3-positive in a tissue micro-array-based, high case number study, whereas 9.2% ofnon-neoplastic liver samples and 16% of preneoplasticnodular lesions were positive.2 In another study byCoston et al5 88% of HCCs were found glypican-3-positive, but focal positivity was occasionally observed inthe surrounding cirrhotic livers as well. Combiningglypican-3 with other markers improves diagnosticaccuracy: Coston et al complemented glypican-3 withCD34 to assess the vascular pattern of the lesions, and a3-marker immunohistochemical panel composed of gly-pican-3, HSP-70, and glutamine synthetase has beenCopyright r 2009 by Lippincott Williams & Wilkins

From the *1st Department of Pathology and Experimental CancerResearch; w2nd Department of Pathology, Semmelweis University,Budapest, Hungary; and zInstitute of Pathology, UniversityHospital, Heidelberg, Germany.

Supported by the grants No. 67925 and 75468 from the HungarianScientific Research Fund (OTKA).

Peter Tatrai and Aron Somoracz are equal contributors.Correspondence: Peter Tatrai, PhD, Research fellow, 2nd Department

of Pathology, Semmelweis University, 93 Ulloi ut, Budapest H-1091,Hungary (e-mail: [email protected]).

ORIGINAL ARTICLE

874 | www.ajsp.com Am J Surg Pathol � Volume 33, Number 6, June 2009

reported to accurately discriminate HGDN and earlyHCC.7

Nevertheless, the quest for reliable, sensitive, andspecific markers of hepatocellular malignancy is still on.Here we propose that the immunohistochemical detectionof agrin, another member of the heparan sulfateproteoglycan family, is a promising novel method forthe identification of malignant hepatocellular lesions. Aswe have described previously,21 agrin is deposited inbiliary and vascular basement membranes of the liver;therefore, it is both a biliary and a vascular marker.Agrin, scarcely present in the normal liver, accumulates incirrhosis due to the formation of ductular reaction andnew blood vessels in the connective tissue septa, and inHCC due to tumor neovascularization. Remarkably,agrin was found highly selective for tumor blood vesselsagainst the sinusoidal walls of both the normal liver andcirrhotic regenerative nodules, raising the possibility thatagrin in microvessels may serve as a marker of HCC.

Therefore, we investigated whether agrin IHC canfacilitate the differential diagnosis of nonmalignant,precancerous and malignant lesions of the liver parench-yma. As agrin is supposed to be a marker of tumor bloodvessels, the discriminative power of agrin IHC wascompared with CD34, a conventional vascular endothe-lial marker. Agrin and CD34 immunostaining patternswere also compared with that of glypican-3 in a selectionof representative and dubious cases.

MATERIALS AND METHODS

Patient’s MaterialOne hundred thirty-eight samples from 86 patients

were enrolled in immunohistochemical evaluation. Speci-mens from liver resections or explanted livers, approxi-mately 2� 2 cm in size, were obtained from the surgicaldepartments of the Semmelweis University, Budapest,Hungary, and from the Institute of Pathology, UniversityHospital, Heidelberg, Germany. Patients with the diag-noses of cirrhosis with or without dysplastic nodules,HCC, HCA, focal nodular hyperplasia (FNH), andBudd-Chiari syndrome were included in the study.

Histopathologic diagnoses were validated by interna-tional experts in liver pathology (P.N., Zs.Sch., P.Sch.).Patient’s data are summarized in Table 1. The study wascarried out with permission from the local ethicscommittees of the institutions involved.

ImmunohistochemistryFor IHC, 5-mm thick tissue sections were depar-

affinized, rehydrated, and subjected to heat-inducedepitope retrieval in Dako Target Retrieval Solution, pH6 (Dako, Glostrup, Denmark) in a boiling pressurecooker for 5 minutes (agrin, CD34, cytokeratin-7), or incitrate buffer, pH 6, in a 98.51C hot water bath for 30minutes (glypican-3). For agrin immunoreactions, enzy-matic treatment with 1mg/mL Pronase E was also carriedout for 1 to 5 minutes (with duration mainly dependingon the age of the specimen). Subsequently, endogenousperoxidase activity was quenched with 10% hydrogenperoxide diluted in methanol. Nonspecific protein bindingwas blocked with 5% bovine serum albumin in phos-phate-buffered saline for 30 minutes. Sections wereincubated at 41C in a humid chamber with the appro-priate primary antibodies overnight. Biotinylated second-ary antibodies (Dako) in a dilution of 1:100 were appliedfor 30 minutes. Signal amplification was carried out eitherwith EnVision system (Dako) in the case of glypican-3, orin 3 subsequent steps: (1) 30 minutes with streptavidin-biotinylated horseradish peroxidase complex (VectastainElite ABC Kit, Vector Laboratories, Burlingame, CA);(2) 10 minutes with biotinylated tyramide in phosphate-buffered saline with 0.03% hydrogen peroxide; (3) 30minutes repeated ABC in the case of agrin, CD34, andcytokeratin-7. Immunorections were visualized usingdiaminobenzidine hydrochloride (DAB Substrate Kit,Vector Laboratories) as chromogen.

All steps were performed at room temperature(251C) unless otherwise specified. Monoclonal anti-human primary antibodies and their dilutions were asfollows: heparan sulfate proteoglycan, clone 7E12,verified as anti-agrin (see Ref. 21), 1:100 (Millipore,Billerica, MA); CD 34, clone QBEnd 10, 1:25 (Dako);

TABLE 1. Clinical and Histopathologic Features

Patient Group N M/F Age (y) Additional Data

Cirrhosis 25 16/9 M: 50.5 (23-63)F: 52 (37-64)

Concomitant hepatocellular lesion (no.): LGDN (7), HGDN (5), sHCC (5), HCC (11)Etiology (no.): chronic hepatitis C (11), PSC (1), Wilson disease (1), no data (12)

FNH 10 0/10 32 (22-46)HCA andadenomatosis

28+2

3/27 M: 53 (51-56)F: 29.5 (22-72)

Degree of histologic atypia (no.): none (16), mild-to-moderate (9), marked to severe (5)

Budd-Chiarisyndrome

1 0/1 24

HCC 27 19/8 M: 59 (31-79)F: 53.5 (21-77)

Histologic grade (no.): I (5), II (14), III (5), not applicable (3): multifocal HCC, FLC,HCCC

Surrounding liver status (no., known etiology): no fibrosis (4), fibrosis (5, 2/5 HCVpositive), cirrhosis (16, 5/16 HCV positive, 1/16 alcoholic), no data (2)

FLC indicates fibrolamellar carcinoma; FNH, focal nodular hyperplasia; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; HCCC, hepatocholangio-cellular carcinoma (with dominantly hepatocytic differentiation); HCV, hepatitis C virus; HGDN, high-grade dysplastic nodule; LGDN, low-grade dysplastic nodule; M/F,male-to-female ratio; PSC, primary sclerotizing cholangitis; sHCC, small HCC.

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glypican-3, clone 1G12, 1:100 (BioMosaics, Burlington,VT); cytokeratin-7, clone OV-TL 12/30, 1:100 (Dako).

Semiquantitative Evaluation of theImmunoreactions

Immunoreactions for agrin and CD34 were eval-uated at low power (� 4 to 5 objective) magnification andgraded on a scale between ‘‘0’’ and ‘‘4+’’. All specimenswere scored by 2 independent observers (P.T., A.S.) witha high degree of concordance (the maximum difference inthe given score was ±1), and disputed cases werereconsidered until consensus was reached.

Grades were defined as follows: ‘‘0’’: none, noimmunostaining is seen in the lesion; it is confined to thestructures within the connective tissue septa such asductular reaction and large blood vessels. ‘‘1+’’: super-ficial, the immunostaining overrides connective tissue/lesion boundaries, but only superficially invades the lesion.‘‘2+’’: intermediate, the immunostaining may deeplyinvade the lesion and form bridges between oppositeconnective tissue septa. In addition, staining inside thelesion noncontiguous with the connective tissue/lesionboundaries may be present. However, the largest con-tinuously stained area is smaller than 1 low-power field(LPF). ‘‘3+’’: incomplete, one or more continuous LPFsinside the lesion show the reaction, but at least 1continuous LPF remains unstained. ‘‘4+’’: complete,immunostaining is present throughout the entire area ofthe lesion, with the largest continuous unstained areabeing smaller than 1 LPF.

Quantitative Evaluation of theImmunoreactions by Digital Morphometry

Slides with agrin and CD34 immunoreactions weredigitized using a MIRAX Midi Scanner (3DHISTECH,Budapest, Hungary). Five randomly selected (but techni-cal quality controlled) view fields at � 10 virtual objectivemagnification were photographed from each slide in theMIRAX Viewer software. Image analysis was performedusing the Leica QWin software (Leica MicrosystemsImaging Solutions, Cambridge, UK). In the case ofnodular lesions not occupying the whole image, an area ofinterest was designated. Ductular reaction surroundingparenchymal nodules was excluded. After manual adjust-ment of color threshold levels to compensate for huedifferences between the samples, pixels corresponding toimmunopositive areas were selected automatically, andthe percent of immunopositive area within the wholeimage or the area of interest was calculated. Results fromthe 5 view fields of the same slide were averaged. Samplegroups were compared by means of Student t test.

RESULTS

Agrin and CD34 Immunostaining Pattern ofCirrhotic Regenerative Nodules, DysplasticNodules, FNH, and Small HCC

Figures 1–3 show representative agrin and CD34immunostainings of benign nodules including cirrhotic

regenerative nodules (Fig. 1), LGDN and HGDN (Fig. 2),as well as of nodules diagnosed as small HCC (Fig. 3).Additionally, the localization of agrin in a cirrhoticnodule is compared with that of cytokeratin-7 in Figure 1.

Typical regenerative nodules were mostly devoid ofagrin immunostaining which in cirrhosis was usuallyconfined to ductular reaction and blood vessels ofconnective tissue septa. However, some agrin immuno-staining was occasionally seen to extend into the interior ofregenerative nodules. Yet, unlike in HCC where agrin wasprimarily localized to the microvessels (see below), agrinwas here associated with cytokeratin-7 rather than CD34(Fig. 1).

Agrin immunopositivity was uniformly lackingfrom LGDN; the immunoreaction appearing within thenodules was associated to inner ductular reaction andblood vessels rather than the sinusoids. As a contrast, inthe few HGDNs examined, heterogeneity in agrin immuno-staining was evident inside the lesions and when compar-ing different lesions, and the strength of agrin reaction wasin parallel with the extent of dysplasia (Fig. 2). SmallHCCs, except for an unusual lesion with massive inflam-matory infiltrate, were strongly immunostained with agrinthroughout the major part of their area (Fig. 3).

Generally, the extent and strength of CD34 immuno-staining correlated with agrin in both benign nodules andsmall HCCs. CD34 immunostaining, however, alwaysoccupied a greater proportion of the lesion area, causingdifferences between benign and malignant nodules to beless evident than in the case of agrin (Figs. 1–3).

In addition to the above, large regenerative nodules(LRNs) of a Budd-Chiari patient, and 10 cases of FNHwere examined as nodular lesions not regarded to beprecancerous. Agrin and CD34 immunophenotype ofthese lesions was found in all relevant aspects similar tocirrhotic nodules (ie, immunoreaction was seen only inbile ductules and blood vessels of the connective tissuesepta but not in sinusoids), hence this is not illustratedseparately.

Immunohistochemical Staining Pattern of HCAand HCC

In ‘‘typical’’ HCAs showing no or very milddysplasia, the parenchyma of the tumor was eithercompletely devoid of agrin (Fig. 4A), or agrin appearedin the sinusoids of periarterial regions characterized bydistinct histologic and immunohistochemical features(Fig. 4C). These regions consisted of smaller, denselypacked cells exhibiting less steatosis than the surround-ing tissue, and their sinusoidal endothelium expressedCD34. The territories immunopositive for CD34 were,however, always broader than agrin-positive areas (Figs.4B and D).

In atypical adenomas with marked to severedysplasia, extensive areas were immunostained with agrin(Fig. 4E). Heterogeneity within these tumors regardingthe degree of dysplasia was common, which was reflectedby the absence or presence of agrin. For example, note thecontrast in the strength of agrin immunoreaction on the

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left and right-hand sides of Figure 4E, and compare withthe increased nucleus to cytoplasm ratio seen on the left-hand side in Figure 4F, top right inset. Unlike agrin,CD34 failed to subtly discriminate between less and moredysplastic areas (Fig. 4F).

HCC was hallmarked by agrin immunostaining ona significant or, as in most cases, the entire area of bothlow-grade and high-grade tumors (Figs. 5A, B). Typi-cally, agrin in HCC was associated with microvesselsonly. However, an additional basement membrane-likeagrin immunostaining around tumor cell groups wasoccasionally also observed and it was apparently inde-pendent from the vasculature (Figs. 5C, D).

Semiquantitative Scoring of IHC and itsCorrelation With Histopathologic Diagnoses

The percentage distribution of agrin immunoreac-tion scores within the sample groups is shown in Figure 6.No score higher than 2+ was observed in the cirrhotic,

FNH, LRN, and LGDN groups, and only occasionally inthe HGDN and HCA groups. In contrast, scores 3+ and4+ prevailed in the sHCC and HCC groups. As apparentfrom Table 2, score 0 was associated with no atypia, andscore 4+ with some degree of atypia in adenomas,whereas score 3+ occurred in perfectly typical cases aswell. Among HCCs with a definable histologic grade, asingle case with score 0 occurred in the well-differentiatedsubgroup, and 4+ tended to be associated with highergrade (Table 2).

On the basis of the above, samples scoring 0 to 2+with agrin were defined as ‘‘benign by agrin IHC’’ orIHC-B, and 3 to 4+ as ‘‘malignant by agrin IHC’’ orIHC-M. Cirrhosis, FNH, and LRNs were not included asbeing obviously benign. Using the histopathologic diag-noses as reference, a specificity of 0.88 and a sensitivity of0.94 was obtained in discriminating benign versusmalignant (Fisher exact test, P<0.0001). Specificity ofthe IHC-based decision could be further improved to 0.93

FIGURE 1. Immunostaining pattern of agrin, CD34, and cytokeratin-7 in cirrhotic regenerative nodules. A typical regenerativenodule with scarce agrin reaction is seen on the top left, whereas the one on the right-hand side exhibits exceptionally strongagrin immunostaining as compared with most regenerative nodules. The lower panels (D–F) are magnified from the area markedin A. Diaminobenzidine chromogen, hematoxylin counterstain. Scale bars: 500 mm (A–C), 100 mm (D–F).



by including the criterion that, in addition to a 3 to 4+agrin score, IHC-M cases must be 4+ for CD34 as well.Here we applied the same logic as Coston et al5 who havedemonstrated that malignant lesions show a completeCD34 immunostaining pattern (that corresponds to 4+in our grading system) in addition to glypican-3positivity. Considering small lesions (LRNs, LGDNs,HGDNs, and small HCCs) only, the combined agrin/

CD34 criteria yielded a sensitivity of 0.87 and a specificityof 1.00 in discriminating benign versus malignant(P<0.0001).

Combined evaluation of agrin and CD34 IHCresulted in decisions discordant with the histopatho-logic diagnosis in 7/103 cases. All the 5 ‘‘false positives’’(ie, diagnosed histologically as benign but classified asIHC-M) were adenomas, including 2 with severe, 1 with

FIGURE 2. Immunostaining pattern of agrin and CD34 in low-grade and high-grade dysplastic nodules (LGDN and HGDN).Agrin immunostaining (A,C,E); CD34 immunostaining (B,D,F). Diaminobenzidine chromogen, hematoxylin counterstain. Scalebars: 2 mm. HGDN indicates high-grade dysplastic nodule; LGDN, low-grade dysplastic nodule.



moderate, and 2 with no atypia. Two ‘‘false negatives’’(ie, diagnosed as malignant but classified as IHC-B)included a single HCC and a single small HCC. The HCCwas a highly differentiated tumor with a monotonouscytologic appearance that developed in a HCV-negative,noncirrhotic liver. An unusual feature of the agrin-negative small HCC was the presence of abundantinflammatory infiltrate (Figs. 3A and B), but whetherand how this finding and weak agrin reaction are relatedis unclear.

Quantitative Evaluation of Agrin and CD34Immunoreactions

Immunoreactions for agrin and CD34 were eval-uated quantitatively using digital morphometry. Thepercentage of immunopositive area within the lesionswas measured and compared across the groups (Fig. 7).In all benign conditions including HCA and HGDN, theaverage percentage of agrin-immunopositive area wassignificantly lower when compared with HCC or smallHCC. Setting the benign versus malignant threshold at5 area%, HCC and HCA could be discriminated by thequantitation of agrin IHC with a sensitivity of 0.80 and a

specificity of 0.89 (Fisher exact test, P<0.0001). Simi-larly, with the threshold set at 5 area% small HCCs andHGDNs were discriminated with a sensitivity of 0.75and a specificity of 1.00 (P=0.007) even in spite of thelow case numbers. CD34, in contrast, failed to differ-entiate quantitatively between HCC and HCA, anddespite significant differences between small HCCs andbenign parenchymal nodules no threshold value forefficient benign versus malignant separation could beestablished.

Comparison of Agrin and Glypican-3 inRepresentative Cases

Agrin, glypican-3, and CD34 immunoreactions onserial sections of selected representative cases are shownin Figure 8. The strength and extent of agrin immuno-staining was comparable in the 3 large HCCs (A, G, M)and 1 small HCC (D) shown here (all score 4+), and itappeared over broad areas (score 3+) in a tumor withuncertain diagnosis that may either be an atypicaladenoma with strong dysplasia (which is the currentdiagnosis) or a well-differentiated HCC (the latter is

FIGURE 3. Immunostaining pattern of agrin and CD34 in small HCCs. Agrin immunostaining (A,C); CD34 immunostaining (B,D).High-power inset in C shows inflammatory infiltrate within the lesion. Diaminobenzidine chromogen, hematoxylin counterstain.Scale bars: 5 mm. HCC indicates hepatocellular carcinoma.



supported by the presence of pseudoacinary structures)(J). CD34 also showed a complete (4+) immunostainingpattern in the malignant lesions (C, F, I, O) and anincomplete (3+) pattern in the dubious case (L). Theextent and intensity of glypican-3 immunostaining, incontrast, was variable in the same cases, ranging from amassive positivity in all tumor cells (B) through modestpositivity observed in all cells (E) or only focally (H) to no

immunoreaction at all (N). A few scattered cells showedweak glypican-3 staining in the dubious case (K).

DISCUSSIONIn this study, we demonstrated that agrin is a

sensitive and specific marker of HCC neovessels thatreliably differentiates benign lesions such as dysplastic

FIGURE 4. Immunostaining pattern of agrin and CD34 in ‘‘typical’’ and ‘‘atypical’’ hepatocellular adenomas. Top right inset in Fshows the different histology on the left and right-hand sides of a connective tissue septum inside the tumor. Diaminobenzidinechromogen, hematoxylin counterstain. Scale bars: 500 mm (A–D), 250 mm (E, F).



nodules and hepatic adenomas from HCC. Althoughagrin is used in this method as a vascular marker, and itmay be useful to complement agrin IHC with CD34immunostaining in ambiguous cases, the latter alone wasfound far less specific for HCC than agrin, indicating thatagrin is more than a mere substitute for long-used,conventional vascular markers. Furthermore, when com-pared with glypican-3 that is the current immunohisto-chemical standard in the differentiation of HCC from benignhepatocellular lesions, agrin immunostaining in certaininstances was more robust and less equivocal to interpret.

Admittedly, agrin immunostaining fails to providethe examiner with a black-or-white picture in whichmalignant lesions are exclusively highlighted. BesidesHCC neovessels, agrin is also associated with benignstructures such as reactive ductules and the walls of majorblood vessels. In our view, however, this can be regardedas an advantage rather than a shortcoming: as these

structures are found in the majority of liver biopsies theymay serve as excellent internal positive controls thatensure the technical fidelity of the immunoreaction.Moreover, in addition to cytokeratin-7, agrin IHC isequally suitable for examining ductular reaction aroundlesions, the presence of which was reported to indicatenoninvasive growth.16

Although the parenchyma of benign nodules wastypically devoid of agrin, some immunostaining wasoccasionally encountered in cirrhotic regenerative no-dules. However, agrin expression here is thought toaccompany the regeneration of the parenchyma fromductular reaction rather than a malignant process.Ductular reaction is a benign proliferation now thoughtto arise from progenitor cells that originally, at restin the healthy liver, reside in the canals of Hering.6 Inthe regenerating liver, these progenitor cells give rise tothe well-known ductules that exhibit biliary epithelial

FIGURE 5. Immunostaining pattern of agrin and CD34 in hepatocellular carcinoma. Only agrin is shown for the tumors in A andB, whereas agrin and CD34 immunostaining patterns of the same tumor area are compared in C and D. Although most of theagrin immunostaining belongs to microvessels (arrows in C; compare with endothelial CD34 pattern in D), a faint basementmembrane-type agrin immunoreaction may occasionally circumscribe pseudoglandular tumor cell structures (arrowheads in C).Diaminobenzidine chromogen, hematoxylin counterstain. Scale bars: 500 mm (A, B), 100 mm (C, D).



phenotype with expression of biliary markers cytokeratin-7 and cytokeratin-19 and deposition of agrin into thebasement membrane. Later on, progression to cirrhosisoccurs and the proliferative capacity of hepatocytescomes near to exhaustion due to long-standing injurysuch as chronic hepatitis, progenitor cells found in theductular reaction may overtake the function of parench-ymal regeneration.12 Reactive ductules were shown to beconnected to groups of cells with an intermediatephenotype (called hepatocyte ‘‘buds’’) that gradually losebiliary markers, for example, they exhibit continuouslyweakening cytokeratin-7 immunopositivity, in the courseof hepatocytic differentiation.8 Similarly, whereas biliaryepithelial-like cells possessing a strongly agrin-positivebasement membrane differentiate into hepatocytes theymay gradually lose agrin expression as well. Thus,

whenever agrin is seen in association with cytokeratin-7-positive cells that form reactive ductules and appear asintermediate phenotype differentiating hepatocytes on themargin of cirrhotic regenerative nodules, its presence isindicative of regeneration rather than malignant trans-formation. The lack of colocalization between agrin andCD34 will further speak against HCC neovessels.

Another exception to the rule that agrin is absentfrom benign hepatocellular lesions was provided byhepatic adenomas where both agrin and CD34 wereobserved not only in atypical tumors but also in somecases with unremarkable histology. Of course, eventhough the average strength and extent of agrin im-munostaining was significantly lower in adenomas whencompared with HCCs, this is a flaw in our method thatshould be accounted for. We hypothesize that theappearance of agrin in adenomas might be related toabnormal perfusion which is a feature that perfectly

TABLE 2. Distribution of Agrin Immunoreaction ScoresAccording to the Degree of Atypia in HCA and HistologicGrade in HCC

Agrin IHC Score

0 1+ 2+ 3+ 4+

HCC (n=30)

Degree of atypiaNone (n=16) 8 2 3 3 0Mildto-moderate (n=9) 1 3 4 0 1Marked to severe (n=5) 0 0 2 2 1

HCC (n=24)

Histologic gradeGrade I (n=5) 1 0 0 3 1Grade II (n=14) 0 0 0 10 4Grade III (n=5) 0 0 0 1 4

HCA indicates hepatocellular adenoma; HCC, hepatocellular carcinoma;IHC, immunohistochemistry.

FIGURE 7. Quantitative evaluation of agrin and CD34 immunoreactions. The bars represent the average percentage ofimmunopositive area and SD in the sample group indicated. P values; a, significant when compared with HCC; b, significantwhen compared with small HCC. HA indicates hepatocellular adenoma; HCC, hepatocellular carcinoma; HGDN, high-gradedysplastic nodule; LGDN, low-grade dysplastic nodule; LRN, large regenerative nodule; sHCC, small HCC.

FIGURE 6. Distribution of agrin IHC scores within the samplegroups. FNH indicates focal nodular hyperplasia; HCA, hepato-cellular adenoma; HCC, hepatocellular carcinoma; HGDN, high-grade dysplastic nodule; IHC, immunohistochemistry; LGDN,low-grade dysplastic nodule; LRN, large regenerative nodule;sHCC, small HCC.



FIGURE 8. Comparison of agrin, glypican-3, and CD34 immunoreactions in selected cases. The arrowheads in K point atscattered, faintly glypican-3-positive cells. Diaminobenzidine chromogen, hematoxylin counterstain. Original magnifications:�20 (A–F, J–O) and �40 (G–I) virtual magnification in MIRAX Viewer.



benign HCAs share with HCCs. HCA is also a lesion withincreased arteriohepatic perfusion, and in areas subjectedto the most intensive hyperperfusion the endothelium oftumoral blood vessels was reported to strongly expressCD34.22 These were the same areas where, thoughspatially more limited than CD34, agrin was present inthe microvasculature of HCA without any accompanyingsign of cellular or architectural atypia. Yet, in contrastwith this incomplete arterialization of HCAs, regularHCCs are fully arterialized: one of the long-established,defining diagnostic features of HCC is the switch fromvenous to arterial blood supply.14 Thus, if agrin behavesas a molecular marker of arterialization, significantdifferences between HCAs and HCCs in the amount ofagrin should remain evident, and this is exactly what wehave observed. For the same reason, whenever strongagrin immunoreaction is encountered in a tumor pre-viously diagnosed as ‘‘atypical’’ adenoma, concern as tothe correctness of the benign diagnosis can be raised.Important work to be done in the future is to correlatethe agrin immunostaining pattern of adenomas with thepostoperative clinical course of the disease and with themolecular characteristics of the tumors, that is, HNF1(a)and (b)-catenin mutation status that, in their turn, alsocorrelate with the histology and the clinical behavior, forexample, the possible malignization of HCA.3,25

In contrast with HCA that showed variable degreeof agrin immunostaining, the classical FNH casesexamined were uniformly agrin-negative. AlthoughFNH is a lesion triggered by vascular malformation andexperiencing arteriohepatic hyperperfusion,9 one couldexpect an agrin immunostaining pattern similar to HCA.However, unlike in HCA that typically lacks intratumoralconnective tissue sheets, blood vessels supplying theparenchymal nodules in FNH radiate from connectivetissue septa. Hence, vascular agrin immunostaining inFNH is largely confined to connective tissue septa andkeeps clear of parenchymal nodules. The consistentabsence of agrin from FNH nodules is also in agreementwith the concept that, as opposed to HCA, FNH is apolyclonal hyperplastic lesion rather than a real tumorand it is thought not to possess malignization potential.17

In addition, worthy of note is that although agrin inthe microvessels was found indicative of HCC, a base-ment membrane immunostaining pattern associated topseudoglandular and trabecular hepatocytic formationswas also observed in strongly dysplastic and malignanthepatocellular lesions. As referred to above, parenchymalregeneration from ductular reaction in cirrhosis impliesbiliary-to-hepatocyte transition normally involving bothloss of expression of biliary markers and disappearanceof the basement membrane. The maintenance of an epi-thelial feature such as a basement membrane may reflect‘‘maturation arrest’’ of pluripotent progenitor cells, oneof the mechanisms proposed for hepatocarcinogen-esis.19,20 As a comparison, well-differentiated cholangio-cellular carcinomas tend to retain an agrin-positivebasement membrane.1 Hence, a basement membrane-likeagrin immunostaining pattern within a hepatocellular

lesion, when it is seen together with agrin positivity ofmicrovessel walls, may confirm that malignant transfor-mation has taken place.

Finally, when proposing a new immunohistochem-ical marker for HCC one has to clarify what additionalbenefit it may confer as compared with the existing andwell-established markers, primarily glypican-3. First,unlike glypican-3 that sometimes identifies only fewscattered cells in a HCC, agrin immunolabeling wasnoted to be extensive in virtually all malignant hepato-cellular lesions. This may be particularly important whenevaluating small specimens such as core biopsies: theprobability of mistaking a malignant lesion for benign ina core biopsy due to sampling error may be decreasedusing agrin. Although this study was entirely based onlarge resection biopsies and the histopathologic diagnoseswere at hand from the beginning, the next logical step isto extend our investigations to core biopsies and evaluatethem in a prospective and blinded fashion. Second, theinternal-positive controls available in the case of agrinthat technically validate the immunoreaction are lackingfor glypican-3 because the latter is not expressed by anynormal adult tissue.

In conclusion, agrin IHC is a valuable tool to assessmultiple features of hepatocellular lesions including thepresence of ductular reaction around nodules, changes invascular structure associated with HCC, and probablyalso aberrant maturation of hepatocytic progenitor cellswhich may lead to HCC. Among these, agrin presentingin the microvasculature was demonstrated to be asensitive and specific indicator of HCC. Therefore, IHCfor agrin, complemented with CD34 when necessary, maygreatly facilitate the discrimination between benign andmalignant hepatocellular lesions.

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Agrin and CD34 Immunohistochemistry for the Discrimination of Benign Versus Malignant Hepatocellular...

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