ANALYSIS OF CHROMOSOMAL INSTABILITY IN PULMONARY OR LIVER METASTASES AND MATCHED PRIMARY HEPATOCELLULAR CARCINOMA AFTER ORTHOTOPIC LIVER TRANSPLANTATION Marine GROSS-GOUPIL 1 , Philippe RIOU 1 , Jean-Franc ¸ois EMILE 2 , Raphae ¨l SAFFROY 1 , Daniel AZOULAY 3 , Isabelle LACHERADE 1 , Aline RECEVEUR 1 , Dominique PIATIER-TONNEAU 4 , Denis CASTAING 3 , Brigitte DEBUIRE 1 and Antoinette LEMOINE 1 1 Service de Biochimie et Biologie mole ´culaire 2 Service d’Anatomie pathologique 3 Centre he ´patobiliaire, Ho ˆpital Universitaire Paul Brousse, INSERM U268, IFR 89 “Biologie inte ´gre ´e de la cellule, Virus et Cancer,” Faculte ´ de Me ´decine Paris-Sud; Assistance Publique-Hôpitaux de Paris, 94804 Villejuif Cedex, France 4 Ge ´nomique Fonctionnelle et Biologie Syste ´mique en Sante ´, FRE 2571, Centre National de la Recherche Scientifique, Villejuif, France To investigate the genetic mechanism of metastatic spread in hepatocellular carcinoma (HCC), we analyzed genomic changes in lung or liver metastases and the corre- sponding primary tumors (83 tumor samples) in 18 patients who underwent orthotopic liver transplantation. We studied the incidence of microsatellite instability (MSI) and loss of heterozygosity (LOH) involving 8 highly polymorphic micro- satellite markers and the polyA tract, Bat26. We also sought alterations of p53 and -catenin gene mutations. High MSI (>30 – 40% of the loci analyzed) was found only in primary tumors (11%), whereas LOH was observed in 50% of primary and in 39% of recurrent tumors. p53 mutations were found in 2 cases of primary HCC but not in the corresponding metas- tases. P53 was overexpressed in 4 primary HCC (22%) and 7 metastases (39%). The percentage of -catenin gene muta- tions was low (6%). Lung metastases retained the D16S402 microsatellite abnormalities observed in the primary tu- mors, whereas recurrent liver tumor did not (p  0.02). In conclusion, LOH and P53 protein overexpression, rather than mutations in the p53 or -catenin genes or MSI, seem to be involved in the spreading of HCC, suggesting the presence of metastasis suppressor genes in the vicinity of the chromo- somal loci in question. © 2003 Wiley-Liss, Inc. Key words: hepatocellular carcinoma; metastasis; p53; microsatel- lite instability; -catenin; loss of heterozygosity The recurrence of cancers remains a major problem despite advances in diagnostic procedures and changes in operative man- agement. The poor outcome may result from an inappropriate choice of treatment at the time of tumor diagnosis. In hepatocel- lular carcinoma (HCC), the overall survival rate after resection is just 35–50%. 1–5 The prognostic criteria are related to the general status of the patient and to the characteristics of the tumor, such as vascular invasion or portal vein tumor thrombus, the number and maximum diameter of nodules and the infiltration of the cap- sule. 6–8 However, these prognostic criteria remain controversial, and neither biologic markers of the tumor nor genetic changes have proved to be efficient prognostic tools for use at the time of diagnosis. About 20 genes carrying somatic mutations have now been identified in HCC. 9 –18 The genetic changes can be divided into at least 4 different pathways, although each pathway appears to be involved in a limited number of tumors. 16,19 –21 These are 1) the p53 pathway involved in the DNA damage response, 2) the reti- noblastoma pathway involved in the control of the cell cycle, 3) the transforming growth factor- (TGF-) pathway involved in growth inhibition and apoptosis, and 4) the antigen-presenting cell (APC)/-catenin pathway implicated in cell-cell adhesion and signal transduction. A recent study by Laurent-Puig et al. 16 de- tected loss of heterozygosity (LOH) on all chromosome arms and mutations in p53, -catenin and axin and suggested 2 main path- ways depending on whether the chromosomal instability was as- sociated with alterations of specific genes or not. The genetic alterations that are the most implicated in the metastatic process of HCC are not yet known. Mutation of the p53 gene and cellular accumulation of the mutated or wild-type P53 protein have been considered poor prognostic factors in patients with HCC; 22,23 ; however, in European HCC the rate of p53 gene mutation is in the vicinity of 15%. 12 On the other hand, we previously reported a significantly lower survival rate without recurrence for the patients with an altered D16S402 microsatel- lite. 24 In our study, we analyzed both matched metastases and primary HCCs from patients who had undergone orthotopic liver transplantation (OLT), which represents an accurate model for studying the metastatic spread of HCC because recurrences in the liver graft cannot be considered to be due to new primary tumors. We followed the 220 patients transplanted for HCC in our insti- tution. Forty-eight of them had recurrent HCC, and 21 underwent curative surgery for metastases. In 18 of them, we analyzed both metastatic and primary tumor gene mutations in the 2 main hepa- tocarcinogenesis pathways (p53 and -catenin) in relation to the presence of microsatellite instability (MSI) or LOH. MATERIAL AND METHODS Study population In all, 1,550 OLTs were performed in our institution between 1975 and 2000, of which 220 were for HCC. The patients did not have extrahepatic metastasis prior to transplantation, and none had intraabdominal metastasis, as assessed by the systematic histologic examination of liver pedicle lymph nodes. Recurrence was observed in 48 of the 220 patients (22%). Most did not undergo surgical treatment; instead, they tended to undergo palliative treatment based on chemotherapy according to the site of the tumor and the severity of the recurrence. Twenty-one patients Grant sponsor: Facult´ e de M´ edecine Paris-Sud; Grant number: UPRES 1596; Grant sponsor: Fondation de l’Avenir ; Grant number: ET9-283; Grant sponsor: Fondation Adrienne et Pierre Sommer, Fondation de France; Grant sponsor: Association pour la Recherche contre le Cancer (ARC); Grant number: 9250; Grant sponsor : Institut du Cancer et d’Immunog´ en´ etique (ICIG, Villejuif). *Correspondence to: Service de Biochimie et Biologie mol´ eculaire, Hˆ opital Universitaire Paul Brousse, 14 avenue Paul Vaillant Couturier, 94800 Villejuif, France. Fax: +33-1-45-59-36-25. E-mail: antoinette.lemoine@pbr.ap-hop-paris.fr Received 27 September 2002; Revised 12 December 2002; Accepted 17 December 2002 DOI 10.1002/ijc.11017 Int. J. Cancer: 104, 745–751 (2003) © 2003 Wiley-Liss, Inc. Publication of the International Union Against Cancer