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Endothelial cell marker expression in dysplastic lesions of the liver: an immunohistochemical study
Journal of Hepatology 34 (2001) 850±857
www.elsevier.com/locate/jhep
Endothelial cell marker expression in dysplastic lesions of the liver: an immunohistochemical study Sophie Frachon 1, GeÂraldine Gouysse 1,2, JeÂroÃme Dumortier 2,3,4, Anne Couvelard 5, Mimoun Nejjari 2, FrancËois Mion 4, FrancËoise Berger 1, Pierre Paliard 4, Olivier Boillot 3,4, Jean-Yves Scoazec 1,2,* 1
Service Central d'Anatomie et Cytologie Pathologiques, HoÃpital Edouard Herriot 69437 Lyon CEDEX 03, France 2 INSERM U45, Lyon, France 3 Unite de Transplantation HeÂpatique, Lyon, France 4 FeÂdeÂration des SpeÂcialiteÂs Digestives, HoÃpital Edouard Herriot, Lyon, France 5 Service d'Anatomie Pathologique, HoÃpital Bichat-Claude Bernard, Paris, France
Backgrounds/Aims: Hepatocellular carcinoma usually contains continuous capillary vessels lacking the differentiation markers speci®c for normal sinusoidal endothelial cells. We therefore aimed to search for alterations in endothelial cell marker expression in precancerous liver lesions. Methods: Expression of the endothelial cell markers CD31, CD34 and BNH9 was analyzed in 138 dysplastic lesions from 40 cirrhotic patients (20 with and 20 without hepatocellular carcinoma). Results: No expression of the three endothelial cell markers was detected in cirrhotic nodules and in non dysplastic regenerative macronodules. The three markers were detected in 29.8% of dysplastic lesions and 47% of hepatocellular carcinomas. At least one marker was detected in 75% of dysplastic lesions and 100% of hepatocellular carcinomas. The three markers were more frequently expressed in areas of small cell than of large cell change (34 vs 10%). No correlation was found with the grade of dysplasia, the occurrence of arterialization and the association with hepatocellular carcinoma. Conclusions: Alterations in the hepatic microcirculation comparable to those observed in hepatocellular carcinoma are present in a signi®cant proportion of dysplastic lesions of the liver and may be indirect markers of the process of liver carcinogenesis. q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Liver carcinogenesis; Liver cell change; Hepatocellular carcinoma; CD31; CD34; BNH9; Angiogenesis; Hepatic microcirculation; Liver pathology
1. Introduction Hepatic sinusoids are highly specialized capillary vessels. Their endothelial lining is fenestrated and devoid of organized basement membrane [1]. Sinusoidal endothelial cells display a distinctive phenotype, characterized by the lack of expression of several normal capillary endothelial cells markers such as CD31 and CD34, and by the expression of speci®c markers, such as CD4, CD14 and CD32 [2,3]. Several studies had concurred to show that the distinctive Received 9 September 2000; received in revised form 7 February 2001; accepted 15 February 2001 * Corresponding author. Fax: 133-472116891. E-mail address: [email protected] (J.-Y. Scoazec).
phenotype of normal liver sinusoidal endothelial cells is usually not expressed in the capillary vessels associated with hepatocellular carcinomas [4±7]. Tumor-associated capillary vessels usually are capillarized, i.e. non fenestrated and limited by a well formed subendothelial basement membrane [4]. Their endothelial lining cells usually express CD31 and CD34, but rarely CD4 and the other functional markers associated with normal sinusoidal endothelial cells [6]. The so-called capillarization of tumor-associated vessels may result either from the development of neo-vessels through a process of tumor angiogenesis or from the alteration of pre-existing sinusoids in response to tumor-induced microenvironmental disturbances.
0168-8278/01/$20.00 q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S01 68-8278(01)0004 9-6
S. Frachon et al. / Journal of Hepatology 34 (2001) 850±857
Emergence of hepatocellular carcinoma is frequently considered the end-stage of a multistep process including the following sequence of lesions: cirrhosis, regenerative macronodules, dysplastic macronodules and early carcinoma. Structural and functional changes in the vascular architecture of the liver are known to occur early in the process of liver carcinogenesis. Arterialization is frequent in dysplastic nodules [8]. Moreover, alterations in the intrahepatic blood ¯ow in precancerous lesions of the liver have been demonstrated by imaging studies [9]. However, few data are available about the structural and functional changes which involve the specialized liver sinusoids during the process of liver carcinogenesis [7,10±12]. In particular, large series are necessary to determine the stage of the process of liver carcinogenesis at which the structural and phenotypic changes characteristic of tumorassociated capillary vessels may be detected and the type of precancerous lesions with which they may be associated. Such data may help to understand the pathogenesis of the vascular changes associated with the emergence of hepatocellular carcinoma and to evaluate their potential interest as markers of the neoplastic transformation. We were therefore prompted to analyze the expression of capillary endothelial cell markers during the various stages of the process of liver carcinogenesis in order to: (a) determine whether alterations of the microvascular phenotype may be detected in precancerous liver lesions, (b) correlate them with the type and grade of change and the occurrence of arterialization, (c) evaluate whether they may be of relevance to the differential diagnosis between high grade dysplasia and early hepatocellular carcinoma. For these purposes, we performed a retrospective immunohistochemical study of a large series of precancerous liver lesions representative of the various types and grades of liver cell change, diagnosed in liver explants obtained during orthotopic liver transplantation procedures. 2. Material and methods 2.1. Study group The study group was composed of patients submitted to orthotopic liver transplantation at HoÃpital Edouard Herriot, Lyon, between 1995 and 1999. Criteria for inclusion were: occurrence of chronic liver disease with cirrhosis and presence of at least one dysplastic lesion at examination of the liver explant, with or without coexisting hepatocellular carcinoma. 81 patients ful®lled these criteria. 40 patients (20 with and 20 without coexisting hepatocellular carcinoma) were randomly selected. There were 29 males and 11 females. The median age was 50 years (range: 30±63 years). The etiology of chronic liver disease was: chronic hepatitis C virus (HCV) infection (17 cases), chronic hepatitis B virus (HBV) infection (three cases), alcoholic cirrhosis (15 cases), primary biliary cirrhosis (one case), hemochromatosis (one case), cryptogenetic cirrhosis (three cases).
2.2. Study material 2.2.1. Examination of liver explants
As previously described [13], liver explants were cut in centimetric
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sections. All macroscopically visible lesions were sampled. Samples were ®xed in 10% formalin and embedded in paraf®n. They were cut into 4 mm sections, and stained with hematoxylin-phloxin-saffron. All sections were analyzed independently by two observers. Selected lesions were then independently classi®ed by three pathologists, unaware of the results of the immunohistochemical study.
2.2.2. Diagnosis and classi®cation
Regenerative nodules were de®ned as nodules measuring more than 8 mm in diameter. The following histopathological characters were recorded: size of the nodule, steatosis, presence of vascular alterations (sinusoidal dilatation, peliotic-like lesions), occurrence of dysplasia. Liver cell changes were classi®ed as large cell or small cell according to the usual cytological criteria [14]. Dysplastic nodules were graded as low or high grade according to the type of liver change and their degree of architectural or cytologic atypia [14]. Arterialization was de®ned by the presence of unpaired arteries, with a distinctive media decorated by an anti-alpha-smooth muscle actin antibody (1A4, Sigma, Saint-Louis, MI, USA).
2.2.3. Study material
At examination of the liver explant, 20 patients had hepatocellular carcinoma and 20 had liver cell change without evidence of HCC. The median age was 56.5 years (^7.5 years) in the ®rst group and 45 years (^6.5 years) in the second. A total of 32 foci of hepatocellular carcinoma were available for study. Eight presented as nodule-in-nodule within dysplastic macronodules. Twenty-six cases were classi®ed as very well differentiated and six as moderately differentiated. A total of 175 macroregenerative nodules was analyzed. One hundred and thirty-eight nodules containing areas of liver cell change were detected. In 91 nodules, only large cell change was detected; 9 out of these 91 nodules were classi®ed as high grade because of marked architectural atypia including presence of scattered pseudoglandular formations. In 47 nodules, small cell change was predominant; it was associated with areas of large cell change in eight cases; 14 nodules presented marked architectural atypia including pseudoglandular formation but retained recognizable portal tract areas. Among the 23 cases of macronodules with marked architectural atypia, 18 showed evidence of arterialization.
2.3. Immunohistochemical study Three endothelial cell markers were assessed: CD31, CD34 and H and Y blood group antigens. The following commercially available antibodies were used: JC70A (CD31, Dako, Glostrup, Denmark), QBend10 (CD34, Immunotech, Marseille-Luminy, France) and BNH9 (H and Y blood group antigens, Immunotech). Serial sections were made from formalin-®xed, paraf®n-embedded liver samples. After antigen unmasking by microwave treatment (5 min in citrate buffer (pH 8)), endogenous peroxidase activity was inhibited. An indirect streptavidin-biotin-peroxidase technique (Dako) was then applied. Peroxidase activity was revealed with diaminobenzidine (Dako). Internal positive controls consisted of the endothelial linings of the vessels of portal tracts or ®brous septa. Negative controls were obtained by omission of the ®rst antibody, replaced either by phosphate-buffered saline or by isotypic immunoglobulins. Results were evaluated as follows. Immunohistochemical staining was considered positive when the endothelial lining of capillary vessels located between hepatocytic plates was distinctly labeled. Immunohistochemical staining was considered homogeneous when it was unevenly distributed along all the capillary vessels present within a precancerous lesion. It was considered heterogeneous in the other cases. Immunohistochemical staining was considered negative when the endothelial lining of the capillary vessels located between hepatocyte plates was unlabeled, whereas internal controls present within the same section were distinctly positive.
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S. Frachon et al. / Journal of Hepatology 34 (2001) 850±857
Fig. 1. CD34 expression in cirrhotic nodules (a) and non dysplastic regenerative macronodules (b). CD34 expression is usually not detected along the sinusoidal vessels in either cirrhotic nodules (a) or regenerative macronodules in the absence of change (b), whereas it is readily detected in the capillary vessels located within ®brous septa. However, rare CD34 positive vessels are visible at the periphery of cirrhotic nodules (arrow) or of macroregenerative macronodules (arrows). Indirect immunoperoxidase followed by Mayer's haematoxylin (original magni®cations: a, £ 100; b, £ 40).
3. Results 3.1. Expression of endothelial cell markers in cirrhotic nodules The endothelial lining of sinusoids in cirrhotic nodules was constantly negative for CD31 and was not labeled by BNH9. Most sinusoidal vessels were negative for CD34. However, CD34 positive vessels were usually detectable at the periphery of cirrhotic nodules, in apparent continuity with septal capillary vessels (Fig. 1a). The labeling for CD31 was restricted to the endothelial cells lining capillaries, arteries and veins in the ®brous septa between cirrhotic nodules and in residual portal tracts. CD34 was constantly detected along capillary vessels in ®brous septa portal tracts. BNH9 monoclonal antibody decorated most capillary vessels in ®brous septa as well as capillaries and portal veins in residual portal tracts. 3.2. Expression of endothelial cell markers in regenerative macronodules without liver cell change The pattern of expression was similar to that observed in cirrhotic nodules. The endothelial lining of intranodular sinusoid vessels was constantly negative for CD31 and
was not labeled by BNH9 monoclonal antibody. Intra-nodular sinusoids were usually negative for CD34. However, positive vessels were usually observed at the periphery of the macronodules or along the ®brous septa extending within the lesion (Fig. 1b). 3.3. Expression of endothelial cell markers in macronodules with liver cell change (Table 1) The capillary vessels associated with areas of liver cell change within macronodules were reactive with CD31 in 42.7% of cases, CD34 in 43.5% of cases and BNH9 in 45.1% of cases (Fig. 2). In macronodules with diffuse Table 1 Expression of endothelial cell markers in regenerative macronodules, dysplastic macronodules and hepatocellular carcinoma Regenerative Dysplastic Hepatocellular macronodules macronodules carcinoma % of cases expressing CD31 CD34 BNH9-related antigens The 3 markers At least 1 marker
0 0 0 0 0
42.7 43.5 45.1 29.8 75
87 86 50 47 100
S. Frachon et al. / Journal of Hepatology 34 (2001) 850±857
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Fig. 2. Expression of endothelial cell markers in small cell (a) and large cell (b) change. CD31 is readily detected along the capillary vessels associated with this nodule of small cell change (a). In contrast, BNH9 monoclonal antibody does not decorate the vessels located within this area of large cell change (b). Indirect immunoperoxidase followed by Mayer's haematoxylin (original magni®cations: a, £ 140; b, £ 140).
liver cell change, the endothelial labeling was evenly distributed all over the lesion. In macronodules containing only scattered foci or nodules of liver cell change, the endothelial labeling was heterogeneously distributed and restricted to the areas of liver cell change. 29.8% of the macronodules with liver cell change contained endothelial cells reactive with the three markers tested (Table 1). Twenty-®ve percent were unreactive with the three markers tested (Table 1). No difference was detected in the histopathological characters of the corresponding lesions (Table 2). We then compared the expression of endothelial cell markers between macronodules containing, respectively, large cell or small cell change (Table 3). The presence of capillary vessels reactive with either CD31, CD34 or BNH9 antibodies was more frequent in lesions of small cell change (respectively, 51, 53 and
56%) (Fig. 2a) than in lesions of large cell change (respectively, 28, 26 and 31%) (Fig. 2b). In the same way, the presence of capillaries reactive with the three markers tested was more frequent in lesions of small cell change (34%) than in lesions of large cell change (10%). Finally, 43% of lesions of large cell change, but only 16% of lesions of small cell change, were unreactive for any of the three markers tested. We could not ®nd any signi®cant difference in the endothelial cell phenotype according to the degree of architectural atypia, the occurrence of arterialization (Table 3) or the association with foci of hepatocellular carcinoma. 3.4. Expression of endothelial cell markers in foci of hepatocellular carcinoma In hepatocellular carcinoma, the endothelial lining of
Table 2 Correlations between endothelial cell marker expression and histopathological features
Expression of: CD31 CD34 BNH9-related antigens The 3 markers None of the markers
Diameter (mm) (mean ^ SD)
Steatosis (% of cases)
Vascular alterations (% of cases)
9.1 ^ 1.7 8.9 ^ 1.9 9.0 ^ 1.5 9.4 ^ 1.8 8.8 ^ 1.7
7 6 8 10 7
5 7 7 6 5
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S. Frachon et al. / Journal of Hepatology 34 (2001) 850±857
Table 3 Correlations between endothelial cell marker expression, type of liver cell change, occurrence of architectural changes, arterialization and association with hepatocellular carcinoma a
% of cases expressing: CD31 CD34 BNH9-related antigens The 3 markers At least one marker a
Cytological features
Architectural changes
Arterialization
HCC
LCC
SCC
absent
present
absent
present
absent
present
28 26 31 10 57
51 53 56 34 84
49.5 53 45 32 72.5
52 55 63 37 79
39 44 39 28 73
62 62 67 36 86
38 40.5 36 25 72
44 43 45 32 79.5
Abbreviations: LCC, large cell change; SCC, small cell change, HCC, hepatocellular carcinoma.
intratumoral capillary vessels expressed CD31 in 87% of cases and CD34 (Fig. 3) in 86% of cases (Table 1). Intratumoral endothelial cells were labeled by BNH9 in 50% of the cases. Labeling was usually homogeneous all over the tumor. When the tumor presented as a nodule-in-nodule, endothelial cell marker expression was closely associated with the carcinomatous nodule (Fig. 3a). In 47% of cases, intratumoral vessels were reactive with the three endothelial cell markers tested. At least one endothelial cell marker was positive in all the cases of HCC tested (Table 1). No close correlation between endothelial cell marker expression and histological differentiation was found (Table 4).
4. Discussion Our results show that several endothelial cell markers characteristic of non-specialized capillary vessels but absent from normal hepatic sinusoids, are undetectable in cirrhotic nodules and in non dysplastic regenerative macronodules, but are frequently expressed by vessels associated with dysplastic macronodules and hepatocellular carcinoma. All three markers tested, CD31, CD34 and BNH9-related antigens, were expressed by sinusoid vessels in 30% of dysplastic lesions, and by tumor-associated vessels in 47% of cases of hepatocellular carcinoma. Moreover, at least one
Fig. 3. Expression of endothelial cell markers in hepatocellular carcinoma. CD34 strongly labels the intra-tumoral vessels of this carcinomatous nodule presenting as a nodule-in-nodule (arrow) within an otherwise non-dysplastic macronodule (a). At a higher magni®cation (b), intra-tumoral vessels readily express CD31. Indirect immunoperoxidase followed by Mayer's haematoxylin (original magni®cations: a, £ 40; b, £ 140).
S. Frachon et al. / Journal of Hepatology 34 (2001) 850±857 Table 4 Correlations between endothelial cell marker expression and histological differentiation in hepatocellular carcinoma
% of cases expressing: CD31 CD34 BNH9-related antigens The 3 markers At least one marker
Well differentiated HCC
Moderately differentiated HCC
100 83 33 50 100
83 88 54 46 100
capillary endothelial cell marker was expressed on sinusoid vessels of 75% of dysplastic lesions and on tumor-associated vessels in all cases of hepatocellular carcinoma examined. Our ®ndings therefore strongly suggest that alterations in the hepatic microcirculation may occur at early stages of liver carcinogenesis, in association with liver cell change or within dysplastic nodules, before the emergence of morphologically identi®able hepatocellular carcinoma. Among the three endothelial cell markers used in our study, two, CD31 and CD34, have been largely employed in previous works. CD31 is directed to PECAM-1, a cellcell adhesion molecule of the immunoglobulin superfamily expressed by most endothelial cells [15]. Previous works have shown that CD31 is not or barely detectable on normal sinusoidal endothelial cells but is overexpressed along sinusoids in various pathological situations, including ®brosis and in¯ammation [3,16]. In the present study, we were unable to detect CD31 in cirrhotic nodules. This apparent contradiction with some previous results from our group is explained by differences in technical conditions. The present study is based on formalin-®xed, paraf®n-embedded liver samples, whereas our previous results [3,16] have been obtained in frozen material. Variations in CD31 expression at the surface of sinusoidal endothelial cells are therefore likely to be of quantitative, rather qualitative, nature. This may hamper the value of CD31 as a marker of vascular changes in the liver. CD34 is a sialomucin expressed by most capillary endothelial cells [17]. Like CD31, CD34 is undetectable on normal sinusoidal endothelial cells. In our study, as in previous ones, including those based on frozen material [5,7,10,16,18,19], CD34 was largely undetectable in cirrhotic nodules and regenerative macronodules. In contrast, CD34 expression could be detected in 43.5% of dysplastic lesions and in 86% of hepatocellular carcinomas. This suggests that, in accordance with previous results [5,7,10,12,18±20], CD34 is a sensitive and speci®c marker of the vascular changes associated with liver carcinogenesis. The third endothelial cell marker used in our study is BNH9 monoclonal antibody, directed to H and Y blood group antigens [21]. In our study, BNH9-related antigens were not detectable in cirrhotic nodules and regenerative
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macronodules but were detectable in 45% of dysplastic lesions and 50% of hepatocellular carcinomas. A more complete characterization of the variations of expression of BNH9 in pathological liver tissue is necessary to assess its potential value as a marker of the vascular changes associated with liver carcinogenesis. The induction of capillary endothelial cell markers that we observed in dysplastic lesions of the liver recalls that observed in hepatocellular carcinoma. The expression of such markers by intra-tumoral vessels has usually been interpreted as a further evidence for the so-called capillarization process associated with hepatocellular carcinoma, ®rst demonstrated by ultrastructural studies [22,23]. Our data, concurring with some previous ones [10,11], strongly suggest that the so-called capillarization process begins early during liver carcinogenesis. They also underline that, despite many morphological similarities, this tumor-associated process may be distinct from the capillarization process observed in cirrhosis, since we were unable to detect comparable phenotypic modi®cations in cirrhotic or regenerative nodules. Two mechanisms may be proposed to explain the alterations of the liver microcirculation associated with liver carcinogenesis. According to a ®rst hypothesis, tumor-associated capillary vessels are neo-vessels produced through a process of tumor angiogenesis [11]. Their occurrence at the early phase of liver neoplasia, as suggested by our results and previous ones [11,12,24], would be in accordance with the current concepts about tumor angiogenesis, since it is usually assumed that pro-angiogenic capacities are acquired early in the development of neoplasia, before the invasive stage. According to an alternative hypothesis, the phenotypic changes detected in tumor-associated vessels may indicate an abnormal differentiation of pre-existing sinusoids. Several lines of evidence suggest that the acquisition and maintenance of the speci®c differentiation of sinusoidal endothelial cells depends on microenvironmental in¯uences [25]. Microenvironmental alterations, such as those resulting from the neoplastic transformation of hepatocytes or from the deposition of tumor-associated extracellular matrix, may therefore be responsible for the loss of the differentiated phenotype of pre-existing sinusoids. The two hypotheses are not mutually exclusive. Their combination may explain the heterogeneity in vascular differentiation that we observed in precancerous lesions of the liver. Neo-vessels coexpressing CD34, CD31 and BNH9 antigens may coexist with altered sinusoid vessels expressing various levels of CD31, CD34 and/or BNH9 antigens in response to microenvironmental changes, including extracellular matrix alterations. In our series, the incidence of microvascular changes was higher in lesions of small cell change than in lesions of large cell change. There is currently much controversy about the biological signi®cance of the two cytological types of liver cell change [8,13,14,26]. However, it is well recognized that the morphological lesions identi®ed as large cell change
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may occur in various settings and, in some cases, may not have a precancerous signi®cance. In contrast, small cell change seems to be more closely associated with liver carcinogenesis. This may explain the higher incidence of microvascular changes in this type of lesion. According to our data, the occurrence of microvascular changes in precancerous lesions of the liver seems not to have any predictive value. We found no correlation between the microvascular phenotype and the grade of dysplasia. In the same way, the incidence of microvascular changes was comparable in patients with or without overt hepatocellular carcinoma. However, the immunohistochemical demonstration of microvascular phenotypic changes may be of diagnostic value. It has been suggested [27±29] that the expression of CD34 by intra-tumoral vessels may be an argument for the diagnosis of hepatocellular carcinoma. However, as shown by our study and previous ones [12], this marker may also be detected in dysplastic lesions. In our experience, the most reliable diagnostic feature is the lack of reactivity of tumor microvessels for capillary endothelial cell markers, which may be a further argument to support the dysplastic nature of a borderline lesion. Further studies, using procedures designed to minimizing biases in histopathologic assessment and improving diagnostic reproducibility, are necessary to verify this possibility. In conclusion, alterations in the hepatic microcirculation comparable to those observed in hepatocellular carcinoma may be detected in a signi®cant proportion of dysplastic lesions of the liver. Vascular alterations may therefore be considered indirect markers of the process of liver carcinogenesis with which they are closely related. Acknowledgements This work was supported in part by grants from ReÂgion RhoÃne-Alpes (Programme Emergence), the Fondation pour la Recherche MeÂdicale and the Hospices Civils de Lyon (Appel d'Offres Recherche Clinique 1997). References [1] Wisse E, De Zangen RB, Charels K, van der Smissen P, McCuskey R. The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse. Hepatology 1985;5:683±692. [2] Scoazec J-Y, Feldmann G. In situ immunophenotyping characterization of endothelial cells of the human hepatic sinusoid: results and functional implications. Hepatology 1991;14:789±797. [3] Scoazec J-Y, Feldmann G. The cell adhesion molecules of human liver sinusoidal endothelial cells. J Hepatol 1994;20:296±300. [4] Kin M, Torimura T, Ueno T, Inuzuka S, Tanikawa K. Sinusoidal capillarization in small hepatocellular carcinoma. Pathol Int 1994;44:771±778. [5] Ruck P, Xiao JC, Kaiserling E. Immunoreactivity of sinusoids in hepatocellular carcinoma. An immunohistochemical study using lectin UEA-1 and antibodies against endothelial markers, including CD34. Arch Pathol Lab Med 1995;119:173±178.
[6] Nakamura S, Muro H, Suzuki S, Sakaguchi T, Konno H, Baba S, et al. Immunohistochemical studies on endothelial cell phenotype in hepatocellular carcinoma. Hepatology 1997;26:407±415. [7] Kimura H, Nakajima T, Kagawa K, Deguchi T, Kakusui M, Katagishi T, et al. Angiogenesis in hepatocellular carcinoma as evaluated by CD34 immunohistochemistry. Liver 1998;18:14±19. [8] Ferrell LD, Crawford JM, Dhillon AP, Scheuer PJ, Nakanuma Y. Proposal for standardized criteria for the diagnosis of benign, borderline, and malignant hepatocellular lesions arising in chronic advanced liver disease. Am J Surg Pathol 1993;17:1113±1123. [9] Maeda T, Adachi E, Kajiyama K, Takenaka K, Honda H, Sugimachi K, et al. CD34 expression in endothelial cells of small hepatocellular carcinoma: its correlation with tumour progression and angiographic ®ndings. J Gastroenterol Hepatol 1995;10:650±654. [10] Park YN, Yang CP, Fernandez GJ, Cubukcu O, Thung SN, Theise ND. Neoangiogenesis and sinusoidal `capillarization' in dysplastic nodules of the liver. Am J Surg Pathol 1998;22:656±662. [11] Park YN, Kim YB, Yang KM, Park C. Increased expression of vascular endothelial growth factor and angiogenesis in the early stage of multistep hepatocarcinogenesis. Arch Pathol Lab Med 2000;124:1061±1065. [12] Roncalli M, Roz E, Coggi G, Di Rocco MG, Bossi P, Minola E, et al. The vascular pro®le of regenerative and dysplastic nodules of the cirrhotic liver: implications for diagnosis and classi®cation. Hepatology 1999;30:1174±1178. [13] Mion F, Grozel L, Boillot O, Paliard P, Berger F. Adult cirrhotic liver explants: precancerous lesions and undetected small hepatocellular carcinomas. Gastroenterology 1996;111:1587±1592. [14] International Working Party. Terminology of nodular hepatocellular lesions. Hepatology 1995;22:983±993. [15] Newman P, Berndt M, Gorski J, White G, Lyman S, Paddock C, et al. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science 1990;247:1219± 1222. [16] Couvelard A, Scoazec J-Y, Feldmann G. Expression of cell-cell and cell-matrix adhesion molecules by sinusoidal endothelial cells in the normal and cirrhotic human liver. Am J Pathol 1993;143:738± 752. [17] Baumheter S, Singer MS, Henzel W, Hemmerich S, Renz M, Rosen SD, et al. Binding of L-selectin to the vascular sialomucin CD34. Science 1993;262:436±438. [18] Cui S, Hano H, Sakata A, Harada T, Liu T, Takai S, et al. Enhanced CD34 expression of sinusoid-like vascular endothelial cells in hepatocellular carcinoma. Pathol Int 1996;46:751±756. [19] Sun HC, Tang ZY, Li XM, Zhou YN, Sun BR, Ma ZC. Microvessel density of hepatocellular carcinoma: its relationship with prognosis. J Cancer Res Clin Oncol 1999;125:419±426. [20] Tanigawa N, Lu C, Mitsui T, Miura S. Quantitation of sinusoid-like vessels in hepatocellular carcinoma: its clinical and prognostic significance. Hepatology 1997;26:1216±1223. [21] Miettinen M, Lindenmayer AE, Chaubal A. Endothelial cell markers CD31, CD34, and BNH9 antibody to H- and Y-antigens: evaluation of their speci®city and sensitivity in the diagnosis of vascular tumors and comparison with von Willebrand factor. Mod Pathol 1994;7:82±90. [22] Ichida T, Hata K, Yamada S, Hatano T, Miyagiwa M, Miyabayashi C, et al. Subcellular abnormalities of liver sinusoidal lesions in human hepatocellular carcinoma. J Submicrosc Cytol Pathol 1990;22:221± 229. [23] Haratake J, Hisaoka M, Yamamoto O, Horie A. An ultrastructural comparison of sinusoids in hepatocellular carcinoma, adenomatous hyperplasia, and fetal liver. Arch Pathol Lab Med 1992;116:67±70. [24] Motoo Y, Sawabu N, Yamaguchi Y, Terada T, Nakanuma Y. Sinusoidal capillarization of human hepatocellular carcinoma: possible promotion by ®broblast growth factor. Oncology 1993;50:270±274. [25] Modis L, Martinez-Hernandez A. Hepatocytes modulate the hepatic microvascular phenotype. Lab Invest 1991;65:661±665. [26] Le Bail B, Bernard PH, Carles J, Balabaud C, Bioulac-Sage P. Preva-
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Endothelial cell marker expression in dysplastic lesions of the liver: an immunohistochemical study Sophie Frachon 1, GeÂraldine Gouysse 1,2, JeÂroÃme Dumortier 2,3,4, Anne Couvelard 5, Mimoun Nejjari 2, FrancËois Mion 4, FrancËoise Berger 1, Pierre Paliard 4, Olivier Boillot 3,4, Jean-Yves Scoazec 1,2,* 1
Service Central d'Anatomie et Cytologie Pathologiques, HoÃpital Edouard Herriot 69437 Lyon CEDEX 03, France 2 INSERM U45, Lyon, France 3 Unite de Transplantation HeÂpatique, Lyon, France 4 FeÂdeÂration des SpeÂcialiteÂs Digestives, HoÃpital Edouard Herriot, Lyon, France 5 Service d'Anatomie Pathologique, HoÃpital Bichat-Claude Bernard, Paris, France
Backgrounds/Aims: Hepatocellular carcinoma usually contains continuous capillary vessels lacking the differentiation markers speci®c for normal sinusoidal endothelial cells. We therefore aimed to search for alterations in endothelial cell marker expression in precancerous liver lesions. Methods: Expression of the endothelial cell markers CD31, CD34 and BNH9 was analyzed in 138 dysplastic lesions from 40 cirrhotic patients (20 with and 20 without hepatocellular carcinoma). Results: No expression of the three endothelial cell markers was detected in cirrhotic nodules and in non dysplastic regenerative macronodules. The three markers were detected in 29.8% of dysplastic lesions and 47% of hepatocellular carcinomas. At least one marker was detected in 75% of dysplastic lesions and 100% of hepatocellular carcinomas. The three markers were more frequently expressed in areas of small cell than of large cell change (34 vs 10%). No correlation was found with the grade of dysplasia, the occurrence of arterialization and the association with hepatocellular carcinoma. Conclusions: Alterations in the hepatic microcirculation comparable to those observed in hepatocellular carcinoma are present in a signi®cant proportion of dysplastic lesions of the liver and may be indirect markers of the process of liver carcinogenesis. q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Liver carcinogenesis; Liver cell change; Hepatocellular carcinoma; CD31; CD34; BNH9; Angiogenesis; Hepatic microcirculation; Liver pathology
1. Introduction Hepatic sinusoids are highly specialized capillary vessels. Their endothelial lining is fenestrated and devoid of organized basement membrane [1]. Sinusoidal endothelial cells display a distinctive phenotype, characterized by the lack of expression of several normal capillary endothelial cells markers such as CD31 and CD34, and by the expression of speci®c markers, such as CD4, CD14 and CD32 [2,3]. Several studies had concurred to show that the distinctive Received 9 September 2000; received in revised form 7 February 2001; accepted 15 February 2001 * Corresponding author. Fax: 133-472116891. E-mail address: [email protected] (J.-Y. Scoazec).
phenotype of normal liver sinusoidal endothelial cells is usually not expressed in the capillary vessels associated with hepatocellular carcinomas [4±7]. Tumor-associated capillary vessels usually are capillarized, i.e. non fenestrated and limited by a well formed subendothelial basement membrane [4]. Their endothelial lining cells usually express CD31 and CD34, but rarely CD4 and the other functional markers associated with normal sinusoidal endothelial cells [6]. The so-called capillarization of tumor-associated vessels may result either from the development of neo-vessels through a process of tumor angiogenesis or from the alteration of pre-existing sinusoids in response to tumor-induced microenvironmental disturbances.
0168-8278/01/$20.00 q 2001 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S01 68-8278(01)0004 9-6
S. Frachon et al. / Journal of Hepatology 34 (2001) 850±857
Emergence of hepatocellular carcinoma is frequently considered the end-stage of a multistep process including the following sequence of lesions: cirrhosis, regenerative macronodules, dysplastic macronodules and early carcinoma. Structural and functional changes in the vascular architecture of the liver are known to occur early in the process of liver carcinogenesis. Arterialization is frequent in dysplastic nodules [8]. Moreover, alterations in the intrahepatic blood ¯ow in precancerous lesions of the liver have been demonstrated by imaging studies [9]. However, few data are available about the structural and functional changes which involve the specialized liver sinusoids during the process of liver carcinogenesis [7,10±12]. In particular, large series are necessary to determine the stage of the process of liver carcinogenesis at which the structural and phenotypic changes characteristic of tumorassociated capillary vessels may be detected and the type of precancerous lesions with which they may be associated. Such data may help to understand the pathogenesis of the vascular changes associated with the emergence of hepatocellular carcinoma and to evaluate their potential interest as markers of the neoplastic transformation. We were therefore prompted to analyze the expression of capillary endothelial cell markers during the various stages of the process of liver carcinogenesis in order to: (a) determine whether alterations of the microvascular phenotype may be detected in precancerous liver lesions, (b) correlate them with the type and grade of change and the occurrence of arterialization, (c) evaluate whether they may be of relevance to the differential diagnosis between high grade dysplasia and early hepatocellular carcinoma. For these purposes, we performed a retrospective immunohistochemical study of a large series of precancerous liver lesions representative of the various types and grades of liver cell change, diagnosed in liver explants obtained during orthotopic liver transplantation procedures. 2. Material and methods 2.1. Study group The study group was composed of patients submitted to orthotopic liver transplantation at HoÃpital Edouard Herriot, Lyon, between 1995 and 1999. Criteria for inclusion were: occurrence of chronic liver disease with cirrhosis and presence of at least one dysplastic lesion at examination of the liver explant, with or without coexisting hepatocellular carcinoma. 81 patients ful®lled these criteria. 40 patients (20 with and 20 without coexisting hepatocellular carcinoma) were randomly selected. There were 29 males and 11 females. The median age was 50 years (range: 30±63 years). The etiology of chronic liver disease was: chronic hepatitis C virus (HCV) infection (17 cases), chronic hepatitis B virus (HBV) infection (three cases), alcoholic cirrhosis (15 cases), primary biliary cirrhosis (one case), hemochromatosis (one case), cryptogenetic cirrhosis (three cases).
2.2. Study material 2.2.1. Examination of liver explants
As previously described [13], liver explants were cut in centimetric
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sections. All macroscopically visible lesions were sampled. Samples were ®xed in 10% formalin and embedded in paraf®n. They were cut into 4 mm sections, and stained with hematoxylin-phloxin-saffron. All sections were analyzed independently by two observers. Selected lesions were then independently classi®ed by three pathologists, unaware of the results of the immunohistochemical study.
2.2.2. Diagnosis and classi®cation
Regenerative nodules were de®ned as nodules measuring more than 8 mm in diameter. The following histopathological characters were recorded: size of the nodule, steatosis, presence of vascular alterations (sinusoidal dilatation, peliotic-like lesions), occurrence of dysplasia. Liver cell changes were classi®ed as large cell or small cell according to the usual cytological criteria [14]. Dysplastic nodules were graded as low or high grade according to the type of liver change and their degree of architectural or cytologic atypia [14]. Arterialization was de®ned by the presence of unpaired arteries, with a distinctive media decorated by an anti-alpha-smooth muscle actin antibody (1A4, Sigma, Saint-Louis, MI, USA).
2.2.3. Study material
At examination of the liver explant, 20 patients had hepatocellular carcinoma and 20 had liver cell change without evidence of HCC. The median age was 56.5 years (^7.5 years) in the ®rst group and 45 years (^6.5 years) in the second. A total of 32 foci of hepatocellular carcinoma were available for study. Eight presented as nodule-in-nodule within dysplastic macronodules. Twenty-six cases were classi®ed as very well differentiated and six as moderately differentiated. A total of 175 macroregenerative nodules was analyzed. One hundred and thirty-eight nodules containing areas of liver cell change were detected. In 91 nodules, only large cell change was detected; 9 out of these 91 nodules were classi®ed as high grade because of marked architectural atypia including presence of scattered pseudoglandular formations. In 47 nodules, small cell change was predominant; it was associated with areas of large cell change in eight cases; 14 nodules presented marked architectural atypia including pseudoglandular formation but retained recognizable portal tract areas. Among the 23 cases of macronodules with marked architectural atypia, 18 showed evidence of arterialization.
2.3. Immunohistochemical study Three endothelial cell markers were assessed: CD31, CD34 and H and Y blood group antigens. The following commercially available antibodies were used: JC70A (CD31, Dako, Glostrup, Denmark), QBend10 (CD34, Immunotech, Marseille-Luminy, France) and BNH9 (H and Y blood group antigens, Immunotech). Serial sections were made from formalin-®xed, paraf®n-embedded liver samples. After antigen unmasking by microwave treatment (5 min in citrate buffer (pH 8)), endogenous peroxidase activity was inhibited. An indirect streptavidin-biotin-peroxidase technique (Dako) was then applied. Peroxidase activity was revealed with diaminobenzidine (Dako). Internal positive controls consisted of the endothelial linings of the vessels of portal tracts or ®brous septa. Negative controls were obtained by omission of the ®rst antibody, replaced either by phosphate-buffered saline or by isotypic immunoglobulins. Results were evaluated as follows. Immunohistochemical staining was considered positive when the endothelial lining of capillary vessels located between hepatocytic plates was distinctly labeled. Immunohistochemical staining was considered homogeneous when it was unevenly distributed along all the capillary vessels present within a precancerous lesion. It was considered heterogeneous in the other cases. Immunohistochemical staining was considered negative when the endothelial lining of the capillary vessels located between hepatocyte plates was unlabeled, whereas internal controls present within the same section were distinctly positive.
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Fig. 1. CD34 expression in cirrhotic nodules (a) and non dysplastic regenerative macronodules (b). CD34 expression is usually not detected along the sinusoidal vessels in either cirrhotic nodules (a) or regenerative macronodules in the absence of change (b), whereas it is readily detected in the capillary vessels located within ®brous septa. However, rare CD34 positive vessels are visible at the periphery of cirrhotic nodules (arrow) or of macroregenerative macronodules (arrows). Indirect immunoperoxidase followed by Mayer's haematoxylin (original magni®cations: a, £ 100; b, £ 40).
3. Results 3.1. Expression of endothelial cell markers in cirrhotic nodules The endothelial lining of sinusoids in cirrhotic nodules was constantly negative for CD31 and was not labeled by BNH9. Most sinusoidal vessels were negative for CD34. However, CD34 positive vessels were usually detectable at the periphery of cirrhotic nodules, in apparent continuity with septal capillary vessels (Fig. 1a). The labeling for CD31 was restricted to the endothelial cells lining capillaries, arteries and veins in the ®brous septa between cirrhotic nodules and in residual portal tracts. CD34 was constantly detected along capillary vessels in ®brous septa portal tracts. BNH9 monoclonal antibody decorated most capillary vessels in ®brous septa as well as capillaries and portal veins in residual portal tracts. 3.2. Expression of endothelial cell markers in regenerative macronodules without liver cell change The pattern of expression was similar to that observed in cirrhotic nodules. The endothelial lining of intranodular sinusoid vessels was constantly negative for CD31 and
was not labeled by BNH9 monoclonal antibody. Intra-nodular sinusoids were usually negative for CD34. However, positive vessels were usually observed at the periphery of the macronodules or along the ®brous septa extending within the lesion (Fig. 1b). 3.3. Expression of endothelial cell markers in macronodules with liver cell change (Table 1) The capillary vessels associated with areas of liver cell change within macronodules were reactive with CD31 in 42.7% of cases, CD34 in 43.5% of cases and BNH9 in 45.1% of cases (Fig. 2). In macronodules with diffuse Table 1 Expression of endothelial cell markers in regenerative macronodules, dysplastic macronodules and hepatocellular carcinoma Regenerative Dysplastic Hepatocellular macronodules macronodules carcinoma % of cases expressing CD31 CD34 BNH9-related antigens The 3 markers At least 1 marker
0 0 0 0 0
42.7 43.5 45.1 29.8 75
87 86 50 47 100
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Fig. 2. Expression of endothelial cell markers in small cell (a) and large cell (b) change. CD31 is readily detected along the capillary vessels associated with this nodule of small cell change (a). In contrast, BNH9 monoclonal antibody does not decorate the vessels located within this area of large cell change (b). Indirect immunoperoxidase followed by Mayer's haematoxylin (original magni®cations: a, £ 140; b, £ 140).
liver cell change, the endothelial labeling was evenly distributed all over the lesion. In macronodules containing only scattered foci or nodules of liver cell change, the endothelial labeling was heterogeneously distributed and restricted to the areas of liver cell change. 29.8% of the macronodules with liver cell change contained endothelial cells reactive with the three markers tested (Table 1). Twenty-®ve percent were unreactive with the three markers tested (Table 1). No difference was detected in the histopathological characters of the corresponding lesions (Table 2). We then compared the expression of endothelial cell markers between macronodules containing, respectively, large cell or small cell change (Table 3). The presence of capillary vessels reactive with either CD31, CD34 or BNH9 antibodies was more frequent in lesions of small cell change (respectively, 51, 53 and
56%) (Fig. 2a) than in lesions of large cell change (respectively, 28, 26 and 31%) (Fig. 2b). In the same way, the presence of capillaries reactive with the three markers tested was more frequent in lesions of small cell change (34%) than in lesions of large cell change (10%). Finally, 43% of lesions of large cell change, but only 16% of lesions of small cell change, were unreactive for any of the three markers tested. We could not ®nd any signi®cant difference in the endothelial cell phenotype according to the degree of architectural atypia, the occurrence of arterialization (Table 3) or the association with foci of hepatocellular carcinoma. 3.4. Expression of endothelial cell markers in foci of hepatocellular carcinoma In hepatocellular carcinoma, the endothelial lining of
Table 2 Correlations between endothelial cell marker expression and histopathological features
Expression of: CD31 CD34 BNH9-related antigens The 3 markers None of the markers
Diameter (mm) (mean ^ SD)
Steatosis (% of cases)
Vascular alterations (% of cases)
9.1 ^ 1.7 8.9 ^ 1.9 9.0 ^ 1.5 9.4 ^ 1.8 8.8 ^ 1.7
7 6 8 10 7
5 7 7 6 5
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Table 3 Correlations between endothelial cell marker expression, type of liver cell change, occurrence of architectural changes, arterialization and association with hepatocellular carcinoma a
% of cases expressing: CD31 CD34 BNH9-related antigens The 3 markers At least one marker a
Cytological features
Architectural changes
Arterialization
HCC
LCC
SCC
absent
present
absent
present
absent
present
28 26 31 10 57
51 53 56 34 84
49.5 53 45 32 72.5
52 55 63 37 79
39 44 39 28 73
62 62 67 36 86
38 40.5 36 25 72
44 43 45 32 79.5
Abbreviations: LCC, large cell change; SCC, small cell change, HCC, hepatocellular carcinoma.
intratumoral capillary vessels expressed CD31 in 87% of cases and CD34 (Fig. 3) in 86% of cases (Table 1). Intratumoral endothelial cells were labeled by BNH9 in 50% of the cases. Labeling was usually homogeneous all over the tumor. When the tumor presented as a nodule-in-nodule, endothelial cell marker expression was closely associated with the carcinomatous nodule (Fig. 3a). In 47% of cases, intratumoral vessels were reactive with the three endothelial cell markers tested. At least one endothelial cell marker was positive in all the cases of HCC tested (Table 1). No close correlation between endothelial cell marker expression and histological differentiation was found (Table 4).
4. Discussion Our results show that several endothelial cell markers characteristic of non-specialized capillary vessels but absent from normal hepatic sinusoids, are undetectable in cirrhotic nodules and in non dysplastic regenerative macronodules, but are frequently expressed by vessels associated with dysplastic macronodules and hepatocellular carcinoma. All three markers tested, CD31, CD34 and BNH9-related antigens, were expressed by sinusoid vessels in 30% of dysplastic lesions, and by tumor-associated vessels in 47% of cases of hepatocellular carcinoma. Moreover, at least one
Fig. 3. Expression of endothelial cell markers in hepatocellular carcinoma. CD34 strongly labels the intra-tumoral vessels of this carcinomatous nodule presenting as a nodule-in-nodule (arrow) within an otherwise non-dysplastic macronodule (a). At a higher magni®cation (b), intra-tumoral vessels readily express CD31. Indirect immunoperoxidase followed by Mayer's haematoxylin (original magni®cations: a, £ 40; b, £ 140).
S. Frachon et al. / Journal of Hepatology 34 (2001) 850±857 Table 4 Correlations between endothelial cell marker expression and histological differentiation in hepatocellular carcinoma
% of cases expressing: CD31 CD34 BNH9-related antigens The 3 markers At least one marker
Well differentiated HCC
Moderately differentiated HCC
100 83 33 50 100
83 88 54 46 100
capillary endothelial cell marker was expressed on sinusoid vessels of 75% of dysplastic lesions and on tumor-associated vessels in all cases of hepatocellular carcinoma examined. Our ®ndings therefore strongly suggest that alterations in the hepatic microcirculation may occur at early stages of liver carcinogenesis, in association with liver cell change or within dysplastic nodules, before the emergence of morphologically identi®able hepatocellular carcinoma. Among the three endothelial cell markers used in our study, two, CD31 and CD34, have been largely employed in previous works. CD31 is directed to PECAM-1, a cellcell adhesion molecule of the immunoglobulin superfamily expressed by most endothelial cells [15]. Previous works have shown that CD31 is not or barely detectable on normal sinusoidal endothelial cells but is overexpressed along sinusoids in various pathological situations, including ®brosis and in¯ammation [3,16]. In the present study, we were unable to detect CD31 in cirrhotic nodules. This apparent contradiction with some previous results from our group is explained by differences in technical conditions. The present study is based on formalin-®xed, paraf®n-embedded liver samples, whereas our previous results [3,16] have been obtained in frozen material. Variations in CD31 expression at the surface of sinusoidal endothelial cells are therefore likely to be of quantitative, rather qualitative, nature. This may hamper the value of CD31 as a marker of vascular changes in the liver. CD34 is a sialomucin expressed by most capillary endothelial cells [17]. Like CD31, CD34 is undetectable on normal sinusoidal endothelial cells. In our study, as in previous ones, including those based on frozen material [5,7,10,16,18,19], CD34 was largely undetectable in cirrhotic nodules and regenerative macronodules. In contrast, CD34 expression could be detected in 43.5% of dysplastic lesions and in 86% of hepatocellular carcinomas. This suggests that, in accordance with previous results [5,7,10,12,18±20], CD34 is a sensitive and speci®c marker of the vascular changes associated with liver carcinogenesis. The third endothelial cell marker used in our study is BNH9 monoclonal antibody, directed to H and Y blood group antigens [21]. In our study, BNH9-related antigens were not detectable in cirrhotic nodules and regenerative
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macronodules but were detectable in 45% of dysplastic lesions and 50% of hepatocellular carcinomas. A more complete characterization of the variations of expression of BNH9 in pathological liver tissue is necessary to assess its potential value as a marker of the vascular changes associated with liver carcinogenesis. The induction of capillary endothelial cell markers that we observed in dysplastic lesions of the liver recalls that observed in hepatocellular carcinoma. The expression of such markers by intra-tumoral vessels has usually been interpreted as a further evidence for the so-called capillarization process associated with hepatocellular carcinoma, ®rst demonstrated by ultrastructural studies [22,23]. Our data, concurring with some previous ones [10,11], strongly suggest that the so-called capillarization process begins early during liver carcinogenesis. They also underline that, despite many morphological similarities, this tumor-associated process may be distinct from the capillarization process observed in cirrhosis, since we were unable to detect comparable phenotypic modi®cations in cirrhotic or regenerative nodules. Two mechanisms may be proposed to explain the alterations of the liver microcirculation associated with liver carcinogenesis. According to a ®rst hypothesis, tumor-associated capillary vessels are neo-vessels produced through a process of tumor angiogenesis [11]. Their occurrence at the early phase of liver neoplasia, as suggested by our results and previous ones [11,12,24], would be in accordance with the current concepts about tumor angiogenesis, since it is usually assumed that pro-angiogenic capacities are acquired early in the development of neoplasia, before the invasive stage. According to an alternative hypothesis, the phenotypic changes detected in tumor-associated vessels may indicate an abnormal differentiation of pre-existing sinusoids. Several lines of evidence suggest that the acquisition and maintenance of the speci®c differentiation of sinusoidal endothelial cells depends on microenvironmental in¯uences [25]. Microenvironmental alterations, such as those resulting from the neoplastic transformation of hepatocytes or from the deposition of tumor-associated extracellular matrix, may therefore be responsible for the loss of the differentiated phenotype of pre-existing sinusoids. The two hypotheses are not mutually exclusive. Their combination may explain the heterogeneity in vascular differentiation that we observed in precancerous lesions of the liver. Neo-vessels coexpressing CD34, CD31 and BNH9 antigens may coexist with altered sinusoid vessels expressing various levels of CD31, CD34 and/or BNH9 antigens in response to microenvironmental changes, including extracellular matrix alterations. In our series, the incidence of microvascular changes was higher in lesions of small cell change than in lesions of large cell change. There is currently much controversy about the biological signi®cance of the two cytological types of liver cell change [8,13,14,26]. However, it is well recognized that the morphological lesions identi®ed as large cell change
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may occur in various settings and, in some cases, may not have a precancerous signi®cance. In contrast, small cell change seems to be more closely associated with liver carcinogenesis. This may explain the higher incidence of microvascular changes in this type of lesion. According to our data, the occurrence of microvascular changes in precancerous lesions of the liver seems not to have any predictive value. We found no correlation between the microvascular phenotype and the grade of dysplasia. In the same way, the incidence of microvascular changes was comparable in patients with or without overt hepatocellular carcinoma. However, the immunohistochemical demonstration of microvascular phenotypic changes may be of diagnostic value. It has been suggested [27±29] that the expression of CD34 by intra-tumoral vessels may be an argument for the diagnosis of hepatocellular carcinoma. However, as shown by our study and previous ones [12], this marker may also be detected in dysplastic lesions. In our experience, the most reliable diagnostic feature is the lack of reactivity of tumor microvessels for capillary endothelial cell markers, which may be a further argument to support the dysplastic nature of a borderline lesion. Further studies, using procedures designed to minimizing biases in histopathologic assessment and improving diagnostic reproducibility, are necessary to verify this possibility. In conclusion, alterations in the hepatic microcirculation comparable to those observed in hepatocellular carcinoma may be detected in a signi®cant proportion of dysplastic lesions of the liver. Vascular alterations may therefore be considered indirect markers of the process of liver carcinogenesis with which they are closely related. Acknowledgements This work was supported in part by grants from ReÂgion RhoÃne-Alpes (Programme Emergence), the Fondation pour la Recherche MeÂdicale and the Hospices Civils de Lyon (Appel d'Offres Recherche Clinique 1997). References [1] Wisse E, De Zangen RB, Charels K, van der Smissen P, McCuskey R. The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse. Hepatology 1985;5:683±692. [2] Scoazec J-Y, Feldmann G. In situ immunophenotyping characterization of endothelial cells of the human hepatic sinusoid: results and functional implications. Hepatology 1991;14:789±797. [3] Scoazec J-Y, Feldmann G. The cell adhesion molecules of human liver sinusoidal endothelial cells. J Hepatol 1994;20:296±300. [4] Kin M, Torimura T, Ueno T, Inuzuka S, Tanikawa K. Sinusoidal capillarization in small hepatocellular carcinoma. Pathol Int 1994;44:771±778. [5] Ruck P, Xiao JC, Kaiserling E. Immunoreactivity of sinusoids in hepatocellular carcinoma. An immunohistochemical study using lectin UEA-1 and antibodies against endothelial markers, including CD34. Arch Pathol Lab Med 1995;119:173±178.
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