The molecular mechanisms of hepatocarcinogenesis recent study on potential familial HCC in Alaskan natives are poorly understood. Only very recently has there excluded mutation of the p53 gene as the responsible genetic been a suggestion of familial hepatocellular carcinoma lesion. 1 This suggests that another tumor-suppressor gene (HCC). We have analyzed the status of the p16 INK4 (MTS1) may be involved in the development of familial HCC. De gene, a cyclin-dependent kinase inhibitor, in 26 patients Souza et al. reported 70% of human HCC exhibited loss of with HCC of different etiologies. Four patients carheterozygosity (LOH) at 6q26-7. 8 They further showed that ried hemizygous germ-line point mutations of the 25% of HCC with LOH contain a point mutation in the man-p16 INK4 (MTS1) gene, suggesting the existence of familial nose 6-phosphate/insulin-like growth factor. 9 HCC involving this gene. The wild-type allele was lost Recently a new tumor-suppressor gene, p16 INK4 (MTS1), in the tumor in 2 of these 4 patients. Three of the patients coding for an inhibitor of cyclin-dependent kinase 4, 10 was carrying a germ-line mutation had non-cirrhosis-associisolated and mapped to chromosome 9p21, a locus frequently ated HCC. No somatic mutations of p16 INK4 (MTS1) were lost in diverse malignancies. 11 Germ-line mutations of the observed in the 26 cases of HCC. The most common so-p16 INK4 (MTS1) gene have been identified in hereditary melamatic alteration of the p16 INK4 (MTS1) gene in HCC was nomas. 12 In addition, the gene is homozygously deleted or de novo methylation, which was detected in 48% of the mutated in uncultured tumors of diverse origin, 13 including cases. Low levels (21%) of p16 INK4 (MTS1) gene allele loss pancreatic adenocarcinomas, 14 melanomas, 12 esophageal carwere observed. Altogether, these results indicate that cinomas, 15,16 and gliomas. 17 Most recently, it has been realteration of the p16 INK4 (MTS1) gene plays an important ported that silencing of the p16 INK4 (MTS1) gene promoter by role in the genesis of HCC. (HEPATOLOGY 1997;25:1376de novo methylation occurs in a variety of tumors. 18 1381.)
We analyzed the 3 exons of the p16 INK4 (MTS1) gene by single-strand conformation polymorphism (SSCP) and DNA Hepatocellular carcinoma (HCC) is one of the most fresequencing in 26 patients with HCC with different etiologiquent human malignancies worldwide. Its incidence shows cal/pathological backgrounds. In addition, we determined the a striking geographic variability, with a high prevalence in methylation status and allele-loss frequency of the populations exposed to aflatoxin B1. Other major etiologic p16 INK4 (MTS1) gene in HCC. Our results suggest that (1) a factors for HCC development are hepatitis B or C virus infecsubset of noncirrhotic HCC cases could be familial, as sugtion and cirrhosis. Recently, the existence of familial inherigested by germ-line mutations of p16 INK4 (MTS1); and (2) sotance of HCC in an Alaskan population has been suggested. 1 matic inactivation of the p16 INK4 (MTS1) gene by methylation The molecular mechanisms involved in hepatocarcinogenor deletion is frequently observed in human hepatocarcinoesis remain poorly understood. The most widely known cangenesis. cer-associated gene in HCC is p53. Somatic mutations in codon 249 have been identified at a high frequency in HCC
PATIENTS AND METHODS
from African and Chinese populations exposed to aflatoxin Patients. Twenty-six patients who underwent partial hepatec-B1. 2,3 However, transfection of a murine p53 gene mutated tomy for HCC in the Departments of Surgery of the University Hospi- at the position corresponding to human codon 249 into hepatals of Bern or Lausanne (Switzerland) were studied. Tumor and tocytes is insufficient to transform hepatocytes. In Western nontumor liver tissue samples were available for histological exami- and other low-aflatoxin B1-exposed countries, somatic p53 nation and DNA extraction. In all cases, the tumor-free liver paren- chyma surrounding the tumor was carefully examined histopatholog-gene alterations are infrequently associated with HCC. ically. Alcohol abuse-related HCC with cirrhosis is the most common Also, hepatitis B-and C-related HCC display a low incitype observed in Switzerland. Because it is rarely treated with surdence of p53 mutations, possibly because of the functional gery, our HCC samples lacking cirrhosis are overrepresented relative to actual incidence. The relevant clinicopathological characteristics are summarized in Table . All patients were white Swiss citizens except patient 11, who was a Bengali refugee. A control group of Abbreviations: HCC, hepatocellular carcinoma; HBX, X protein encoded by hepatitis B 159 individuals without manifest neoplasms was also analyzed for virus; LOH, loss of heterozygosity; MTS1, multiple tumor suppressor 1; SSCP, single-strand p16 INK4 (MTS1) germ-line alterations. conformation polymorphism; PCR, polymerase chain reaction; bp, base pairs; CEPH, Centre DNA Extraction. The tissues analyzed were either frozen or formad'Etude du Polymorphisme Humain.