The poor prognosis of hepatocellular carcinoma (HCC) is partly the result of the high rate of recurrence that is caused either by intrahepatic metastasis (IM) or independent multicentric occurrence (MO). For convenience, discrimination of IM and MO is based on pathological findings, but reliable parameters are not sufficiently established. In the case of hepatitis B virus (HBV)-associated HCC, molecular discrimination of IM from MO can be achieved by comparison of integrated HBV DNAs. However, Southern blotting cannot be used for this purpose when one tumor is saved in frozen form and the other is in paraffin-embedded form. To solve this problem, we employed polymerase chain reaction (PCR) assays to confirm the clonality of primary and recurrent tumors. From the frozen tissue, we determined the junction between the integrated HBV and flanking genomic DNA by molecular cloning, and checked the existence of an identical junction in the DNA of paraffinembedded tissue by PCR. Using this method, as well as Southern blotting, we proved in 6 of 8 patients that two nodular HCC lesions resected metachronously or simultaneously were caused by MO, while the remaining 2 cases were caused by IM. In 1 IM case, band patterns between two HCCs detected by Southern blotting were not identical. (HEPATOLOGY 1999;29:1446-1452.)
Hepatocellular carcinoma (HCC), one of the most common cancers in many parts of the world, is the third-leading cause of death from malignancy in Japan. In 1995, HCC was responsible for about 30,000 deaths, and the number of deaths continues to increase in Japan. 1 Improvement in imaging modalities and strict follow-up of patients with hepatitis B virus (HBV) or hepatitis C virus, including measurement of serum tumor markers, allows early detection of HCCs. In spite of technical advances in the treatment of HCC, such as surgical resection, percutaneous ethanol injection therapy, and microwave coagulation therapy, the longterm survival rate is still low. One of the major reasons for this poor prognosis is the high rate of recurrence, which is 20% to 40% within 1 year, and about 80% in 5 years after curative operation in Japan. 2-5 HCC recurrence may be the result of intrahepatic metastasis (IM) or independent multicentric occurrence (MO). Discrimination between the two is important not only for the study of hepatocarcinogenesis, but also for determination of therapeutic strategies. Some groups have reported that HCC with IM recurs earlier and has a poorer prognosis than its MO counterpart. Aggressive therapy may not be warranted in cases with widespread metastatic dissemination, but in cases with MO, intervention should be taken within the limits of liver functional reserve. The diagnosis of IM or MO is mainly based on pathological findings as reported by the Liver Cancer Study Group of Japan 7 with modifications, or on the analysis of HBV-DNA integration by Southern blotting in cases of HBV-associated HCC. The pathological criteria for MO reflect the multistep nature of HCC development. Metastasis does not appear to occur from atypical adenomatous hyperplasia or very well-differentiated HCCs (Edmondson' s grade I) with maintenance of a relatively normal trabeculae structure, so that these cases can be considered to be primary de novo cancers. In addition, moderately to poorly differentiated lesions with elements of well-differentiated HCC at their margin may also be considered as MO. However, we often encounter cases for which clear distinction is impossible based on pathological findings alone.
Comparison of integrated HBV DNA in primary and recurrent HCCs by Southern blotting is a more accurate method to discriminate IM from MO. However, Southern blotting cannot always be used, because in most cases, the primary HCC, obtained several years before recurrence, is stored only in paraffin-embedded blocks. To overcome this difficulty, we employed polymerase chain reaction (PCR) assays to confirm the clonality of the primary and recurrent tumors. By PCR and Southern blotting, we here showed that 6 of 8 cases of the recurrence or the synchronous plural occurrence of HCC were caused by MO, and that, in 2 cases, the recurrent tumors were the result of IM. We could not distinguish between MO and IM origins of HCC lesions in 4 of the 8 cases based on pathological findings. In 1 IM case, Abbreviations: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; IM, intrahepatic metastasis; MO, independent multicentric occurrence; PCR, polymerase chain reaction.