Hepatitis B virus (HBV), a virus with known carcinogenic potential, integrates into cellular DNA during long-term persistent infection in man. Hepatocellular carcinomas isolated from viral carriers often contain clonally propagated viral DNA integrations. As small chromosomal deletions are associated with several types of carcinomas, the occurrence of chromosomal deletions in association with HBV integration in hepatocellular carcinoma was studied. HBV integration was accompanied by a deletion of at least 13.5 kilobases of cellular sequences in a human hepatocellular carcinoma. The viral DNA integration and deletion of cellular sequences occurred on the short arm of chromosome 11 at location 11p13-11p14. The cellular sequences that were deleted at the site of HBV integration were lost from the tumor cells, leaving only a single copy of the remaining cellular allele.

Normal liver and hepatocellular carcinoma (HCC) genotypes were compared at loci on most of the human chromosomes with probes that detect restriction fragment length polymorphisms. Six of fourteen tumors exhibited loss of heterozygosity of one or more markers on 11 p. Ten patients were informative for loci on 13q, and 5 of these 10 exhibited loss of heterozygosity for one or more of the 13q markers. Altogether, 9 of the 14 patients showed loss of a polymorphic allele for one or more loci on either 11p or 13q. A survey of loci on 16 additional chromosomes indicated that the deletions were not due to a general loss of heterozygosity in HCCs. Quantitative densitometry showed that each of the 10 deletions resulted in hemizygosity (no reduplication) of the remaining allele in tumor tissue. In contrast to hereditary embryonal tumors, in which reduplication of the remaining chromosome is the rule, simple deletion appears to be the primary mechanism responsible for the loss of heterozygosity in these adult, nonhereditary HCCs. These data show that HCCs arising in hepatitis B virus carriers are a genetically heterogeneous group of tumors, some of which may arise through 13q alterations, some through 11p alterations, some with both chromosomes altered, and some with both intact.

One in 10,000 children develops Wilms' tumour, an embryonal malignancy of the kidney. Although most Wilms' tumours are sporadic, a genetic predisposition is associated with aniridia, genito-urinary malformations and mental retardation (the WAGR syndrome). Patients with this syndrome typically exhibit constitutional deletions involving band p13 of one chromosome 11 homologue. It is likely that these deletions overlap a cluster of separate but closely linked genes that control the development of the kidney, iris and urogenital tract (the WAGR complex). A discrete aniridia locus, in particular, has been defined within this chromosomal segment by a reciprocal translocation, transmitted through three generations, which interrupts 11p13. In addition, the specific loss of chromosome 11p alleles in sporadic Wilms' tumours has been demonstrated, suggesting that the WAGR complex includes a recessive oncogene, analogous to the retinoblastoma locus on chromosome 13. In WAGR patients, the inherited 11p deletion is thought to represent the first of two events required for the initiation of a Wilms' tumour, as suggested by Knudson from epidemiological data. We have now isolated the deleted chromosomes 11 from four WAGR patients in hamster-human somatic cell hybrids, and have tested genomic DNA from the hybrids with chromosome 11-specific probes. We show that 4 of 31 markers are deleted in at least one patient, but that of these markers, only the gene encoding the beta-subunit of follicle-stimulating hormone (FSHB) is deleted in all four patients. Our results demonstrate close physical linkage between FSHB and the WAGR locus, suggest a gene order for the four deleted markers and exclude other markers tested from this region. In hybrids prepared from a balanced translocation carrier with familial aniridia, the four markers segregate into proximal and distal groups. The translocation breakpoint, which identifies the position of the aniridia gene on 11p, is immediately proximal to FSHB, in the interval between FSHB and the catalase gene.

Hepatocellular carcinoma is the culmination of a series of genetic events which progressively alter the phenotype of a hepatocyte toward malignancy. Hepadnaviral DNA integrations are agents of genetic change which can promote the process of hepatocarcinogenesis. We previously characterized episomally derived duck hepatitis B virus (DHBV) integrations in LMH-D2 cells that replicate wild-type DHBV. In an effort to understand how integrations function as agents of progressive genetic change, we have studied integrations of DHBV DNA in three lineages of LMH-D2 cells through three generations of subclones. Our data have established several features of the integration process. First, single and multiple integrations occur continuously through successive cell generations. Second, the integration frequency can vary dramatically in subclones of the same cell line. Third, integrations can be lost from successive generations of cells and loss of an integration can be accompanied by loss of cellular DNA associated with the integration. Finally, certain subclones which acquire greater plating efficiency have been distinguished by unique new integration patterns. These results provide a basis for DHBV integrations to function as activators of protooncogenes, as well as agents of the loss of tumor suppressor genes during hepatocellular carcinogenesis.