Determination of the clonal origin of multiple human hepatocellular carcinomas by cloning and polymerase chain reaction of the integrated hepatitis B virus DNA

Abstract

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. 2,6 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, 6,[8][9][10][11] or on the analysis of HBV-DNA integration by Southern blotting in cases of HBV-associated HCC. 8,[12][13][14][15][16][17][18] The pathological criteria for MO reflect the multiste...

Cytokeratin 19 expression in hepatocellular carcinoma predicts early postoperative recurrence

“…To elucidate the origin of tumor recurrence, further studies based on clonal genetic analysis are needed. [28][29][30] However, it is too difficult to perform such analysis of all recurrent tumors.…”

Primary liver resection and salvage transplantation or primary liver transplantation in patients with single, small hepatocellular carcinoma and preserved liver function: An outcome-oriented decision analysis

“…Where evidence was insufficient, however, we deliberately chose figures penalizing the strategy of primary resection and salvage liver transplantation that we wanted to test. Accordingly, we attributed to salvage transplantation a 5 year survival of 60%, assuming that in some cases recurrence after resection may be intrahepatic metastases, 45 and that these tumors may have increased potential for distant spread. In a recent report, however, recurrent disease developed in only 1 of 12 patients transplanted for recurrent HCC after resection, 46 and it is possible that the survival figures will need adjusting in favor of salvage transplantation.…”

Clinicopathologic Characteristics and Prognoses for Multicentric Occurrence and Intrahepatic Metastasis in Synchronous Multinodular Hepatocellular Carcinoma Patients

“…As reported, recurrent HCC has two origins: multicentric occurrence (MO) and intrahepatic metastasis (IM) (Yamamoto et al, 1999). The MO recurrence of HCC is new and different from the primary lesion, but for IM, the recurrence is derived from the primary tumor.…”

“…Patients with MO have better outcomes than those with IM (Yasui et al, 1997;Huang et al, 2012). Many studies (Oda et al, 1992; Liver Cancer Study Group of Japan, 1997;Yamamoto et al, 1999;Chen et al, 2000;Nomoto et al, 2002;Okamoto et al, 2006;Nomoto et al, 2007;Li et al, 2008) have examined the differentiation of the origins of recurrent HCC, also known as metachronous multiple HCC, because the original type is important for the preoperative assessment, determining the treatment modality and predicting patient prognosis. The same problem also appears in synchronous multinodular HCC patients, but few studies have examined this population.…”