Dna Ploidy Analysis of Hepatic Preneoplastic and Neoplastic Lesions in Woodchucks Experimentally Infected With Woodchuck Hepatitis Virus

Abstract

We analyzed the DNA ploidy and the nuclear size of hepatocytes within hepatocellular carcinoma, putative preneoplastic (clear cell and basophilic foci) and adjacent non-neoplastic liver in 30 woodchucks neonatally infected with the woodchuck hepatitis virus. In livers from control woodchucks, in clear cell foci and in most chronic portal hepatitis, the hepatocytes were diploid, with less than 10% tetraploid cells. Aneuploid peaks were found in 50% of the livers with chronic active hepatitis, in 63% of basophilic foci and in 90% of hepatocellular carcinoma. Within the same tumor, aneuploid peaks with different DNA indices were observed frequently, indicating heterogeneity of tumor. S-phase was always elevated, indicating an increased rate of proliferation. Aneuploid cells had nuclei that were larger than those of control liver cells. In some basophilic foci and in some livers with chronic active hepatitis, abnormal DNA was demonstrated before the development of hepatocellular carcinoma, suggesting that these may be populations of hepatocytes at risk of neoplastic transformation. (HEPATOLOGY 1994;20:21-29.)Woodchucks may be infected with the woodchuck hepatitis virus (WHV) that causes chronic hepatitis and hepatocellular carcinomas (HCC) without cirrhosis (1,2). Certain lesions such as basophilic or hyperplastic nodules, clear cell foci and nodules have been described associated with or preceding HCC (3-6), but little is known about the evolution of these lesions. 31/1/66809Foci of altered hepatocytes appear during hepatocarcinogenesis induced by chemicals and they have been considered precursors of carcinomas based on morphological, enzymatic and biochemical studies (7-11). These foci may display abnormal DNA content (12, 13) and may constitute a cell population at risk of neoplastic progression. Little is known, however, regarding the biological behavior of altered hepatocellular foci observed during viral hepatocarcinogenesis. A recent report indicates that focal enzymatic alterations emerging in WHVinfected woodchucks may be closely linked to neoplastic transformation of hepatocytes (14).DNA ploidy studies have become an important method in cancer research. They can provide valuable information and be useful in assessing tumor aggressiveness and prognosis ( 15-2 1).To better understand the biological characteristics of the putative preneoplastic and neoplastic lesions in viral hepatocarcinogenesis, we have analyzed by image analysis and flow cytometry the DNA ploidy and the nuclear size of the putative preneoplastic lesions and of HCC of woodchucks experimentally infected with WHV injection. MATERIALS AND METHODSThirty-seven formalin-fixed, paraffin-embedded liver biopsy blocks from 30 woodchucks were obtained from the New York State College of Veterinary Medicine, Ithaca, New York. These woodchucks had been inoculated subcutaneously during the first 3 days of life with 100 p,l of a 10-dilution of the WH7-PI pool infected with WHV. Woodchuck hepatitis surface antigen (WHsAg) and antibodies were detected in the...