Expression of cadherins in primary liver tumors

$82 HEPATOCELLULAR CARCINOMA I P2 c91220 1

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E X P R E S S I O N O F C - M Y C A N D H I S T O N E H32 T R A N S C R I P T S DURING HEPATOCARCINOGENESIS IN W H V - M Y C T R A N S G E N I C MICE P. Liu. C.A. Renard, G. Feldm~nn, M.AT Buendia, D. Bernu~u. Laboratoire de Biologie Cellulaire,I N S E R M U 327,Facult~ de M6decine X. Bichat, and Unit~ de Recombinaison et Expression G6n~ique, INSERM U 163, InstitutPasteur, Pats France. Transgenic mice carrying the c-myc oncognne under the control of woodchuck hepatitis virus ( W H V ) D N A sequences develop hepatocarcinomas at a high frequency. This model is particular by a temporally limited expression of the transgene detectable by Northern blotting only during the first 3 neonatal weeks and in liver tumors developing after 8-12 months of life.By using in situ hybridization,we explored the expression patterns of the transgene at all stages of the carcinogenic process. Specific questions adressed were I) is the c-myc transgene expression totallyextinguished during the pretumoral period ? and 2) is there a correlation between c-myc oncogene activation and liver cell proliferation ? In 10 days-old mouse liver sections, c-myc transgene transcriptswere detected at high levels over all hepatocytes, without any zonal distributionand 30 % of cellsexpressed histone H32 m R N A , a marker of S-phase cells.In 2 months-old mouse liversections, no histological abnormality was visible, except for the presence of dysplastic hepatocytes, isolatedor in small clustersaround centrilobular veins. N o significantsignal for c-myc transgene transcriptswas visible, especiallyover these dysplastichepatocytes. A very small number (0.5%) of hepatic cells expressed histone H32 m R N A . At the tumoral stages, aden0mas and hepatocarcinomas expressed diffusely c-myc transgene m R N A , at a level higher than the surrounding peritumoral tissue,and 916 % of the tumoral ceilsexpressed histone H32 m R N A , contrasting with a moderate number of proliferativecellsin the peritumoral tissue(I-5%). In summary, c-myc transgene expression is totallyextinguished during the pre-tumoral period, despite the appearance of dysplastic lesions. There is a correlation between the level of c-myc expression and cell proliferation.These data suggest that c-myc overexpression is important for the development and maintenance of a transformed phenotype.

EXPRESSION OF CADHERINS IN PRIMARY LIVER TUMORS I Kozvraki. J-Y Scoazec. J-F Fleiou. A D'Errico. P Bedossa. M Fiorentino. A-F

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ADDITIVE PREDICTIVE VALUE OF LIVER CELL DYSPLASIA IN DEFINING A HIGH RISK GROUP FOR HEPATOCELLULAR C A R C I N O M A (HCC) AMONG WESTERN PATIENTS WITH CIRRHOSIS. N.G~nne, Cl.Chastamz (1`1. D.Paleron. JC.Trinchet, M.Sibonv (2"1.C.Munz (2). P.Callard (2). M.Beano_rand. Service d'H6palo-gastroent6rologie; Service d'Anatomo-pathologie (2) HOpital Jean Verdier, 93143 Bondy Cedex F; D6partment de Biostatistique et d'Informatique M6dicalc, H0pital SaintLouis, Paris-F (1). The aim of this study was to identify among unselccled Western patients with cirrhosis a group at high risk for HCC, on the basis of a limited number of variables usable for a clinical purpose. In 200 patients hospitalized for cirrhosis from January 1987 to January 1990 and pros~ctively screened for HCC until death or November 1992, 40 developed HCC. We recorded in these patients the predictive value of 24 variables for the occurrence of HCC. Variables noted at enrollement were analysed by univariate (Kaplan-Meier method, log-rank test) and multivariate analysis (Cox model). Independant clinical and biological prediclive variables were : age ->55 years (p < 10"5), male sex (p < 10"5), prothrombin activity <70% (p = 0.02) and serum bilirubin _>17 I-tm/L (p = 0.02). A predictive score including this 4 variables allowed to define 2 groups of patients at low (n = 104, 4 HCC) and high risk for HCC (n = 96, 36 HCC) in which cumulative incidences (CI) of HCC at 3 years were respectively 4% and 40%. The study of histological variables for 151 patients in whom liver biopsy was performed at enrollement showed that liver cell dysplasia : 1) was an independant predictive variable for the occurrence of HCC (p = 0.035) 2) had an additionnal predictive value in patients with high score (p = 0,001) and 3) helped to define a group at very high risk for HCC (CI at 3 years = 70%). We concluded that in patients with cirrhosis clinical and biological variables help to define a group of patients at high risk for HCC, and lhat in these patients liver biopsy is helpfull to detenninate a group of patients at an even higher risk.

Brineuier. W Grieioni. A-M Mancini. G Feldmarm. INSERM U327, Paris, France; H6piud Beaujon, Clichy, France; H6pital de Bic&tre, France; Policlinico S. Orsola, Bologna, Italy. Cadherins arc specialized membrane proteins involved in the maintenance of intercellular adhesion. Alterations in cadherin expression are frequent in malignant tumors and might contribute to local invasion. Little is known about cadherin expression in the normal human liver and in primary liver tumors. We analyzed by immunohismchemistryand Western blotting (WE) the expression of cadherins by normal liver epithelial cells, and in a large series of primary liver tumors. The study group consisted in: (a) normal liver tissue samples (15), (b) benign liver tumors: focal nodular hyperplasia (25), regenerative nodular hyperplasia (2), solitary liver cell adenomas (9), (c) primary malignant liver tumors: hepatocellu]ar carcinomas (HCC) (65) and cholangiocarcinomas (CHC) (9). The monoclonal antibodies used were: (a) 6F9 and HCED-1, specific for Ecadherin, expressed by most epithelial cells, (b) GC-4, directed against members of the N-cadherin family, expressed by neural cells and some epithelial cell subsets. In normal human liver, liepatocytes expressed both E-cadherin, with a main form at 130 kD and a minor form at 145 kD, and GC4-reactive cedherin, with a main form at 124 kD and a minor form at 137 kD. Bile duct cells expressed only E-cadherin. Benign liver tumors were characterized by the constant coexpression of both E- and GC4-reactive cadherins. WB analysis showed a pattern comparable to that of normal liver. Well-differentiated HCC (11 cases) always coexpressed E- and GC4-reective cadherins. Qualitative alterations were detected by WB in 4 out of the 5 cases tested (predominance of the high molecular weight forms, presence of additional forms of low molecular weight). In moderately and poorly-differentiated HCC (54 cases), 4 immunohistochemica.l patterns were observed: (it) coexpression of E- and GC4-reective cedlierins (17 cases), (b) expression of Ecedherin only (11 cases), (c) expression of GC4-reactive cadherin only (14 cases), (d) complete loss of cadharin expression (12 cases). When cadherin expression was retained, qualitative alterations were observed: (a) frequent loss of polarization at coil surface, (b) abnormal W'B pattern. In CHC, E-cadherin was detected in 7 cases out of 9. No qualitative alteration was detected by WE. GC4-reactive cedherin was always negative. In conclusion, (a) normal hepatocytes and bile duct cells express distinctive combinations of cadherins, (b) the normal pattern of cadherin expression is retained in benign liver tumors, (c) quantitative and qualitative changes in cadherin expression are detected in a large proportion of primary malignant liver tumors.

STUDY OF PROLIFERATION INDEX USING PCNA IN FOCI OF SMALL/LARGE LIVER CELL DYSPLASIA (LLCD/SLLD), CIRRHOTIC NODULES AND HEPATOCELLULA.R CARCINOMA. M.Demonakou, A.Rayner, Roger Williams, B.Portmann. Institute of Liver Studies, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 9RS, U.K. The precursor lesions of hepatocellular carcinoma (HCC) are not well defined in man. LLCD, SLCD, atypical adenomatous hyperplasia (AAH) or borderline nodules (BN) have been variably considered as potential candidates. Using PCNA-PCIO on paraffin sections we have studied the proliferative index of LLCD and SLCD foci, nonneoplastic and neoplastic nodules identified in 150 cirrhotic livers of diverse aetiology removed at the time of transplantation. Five donor's livers wer.= used as normal controls (NL). LLCD was found in 8%(12/150), SLCD in 14.7%(22/150) and one or more nodules (N = 38) in 14.7 %(22/150) of the livers. These 38 nodules included 14 HCC, 19 AAH/BN, 1 adenoma and 4 multiacinar cirrhotic nodules (MCN). The mean values of PCNA positive nuclei per 1000 cells counted were: NL:0 < LLCD:I < adenoma:2 < MCN:2 < SLCD: 12 < AAH/BN: 14 < HCC:73. NL and LLCD had similarly low values which were not significantly different from those obtained in the single adenoma and in MCN. In contrast results in SLCD were markedly and significantly higher than in the NL (p < 0 . 0 5 ) , close to values obtained in AAH/BN and significantly lower than those of, HCC (p