Clonality of large regenerative nodules in liver cirrhosis

The role of the large regenerative nodule (RN) in hepatocarcinogenesis is not clear, although the incidence of hepatocellular carcinoma (HCC) is high in cirrhotic liver. This study was aimed at clarifying the preneoplastic nature of large RN without atypia. We analyzed the clonality of HCCs and large RNs, ranging in size from 0.6 to 1.2 cm, of cirrhotic liver by X-linked human androgen receptor (HUMARA) gene assay, using the principle of random X chromosome methylation and inactivation in females. Eleven cases of HCC and five cases of large RN without atypia from ten female patients were selected. All HCCs, large RNs and paired non-tumorous tissue from adjacent liver were selectively microdissected from deparaffinized hematoxylin and eosin stained slides. Genomic DNA was isolated and digested with Hha I. Polymerase chain reaction (PCR) amplification of the HUMARA gene was performed using a PCR mixture containing [alpha-32P]-dCTP. The PCR products were separated by gel electrophoresis and analysed by autoradiography. HUMARA was informative in nine out of ten female patients. In the informative 10 HCCs from nine patients, 9 HCCs were monoclonal and one case was polyclonal. The HCC case that showed polyclonality contained many inflammatory cells in the tumor. All of the large RNs were polyclonal. No allelic loss of chromosome 18q was present in the large RNs in constrast to the 3 out of 7 HCCs, which showed allelic deletion in chromosome 18q. We conclude that all or most of the cells composing the large RNs without atypia are polyclonal and the size of a nodule may not be important in hepatocarcinogenesis. This clonality assay may be informative for the differentiation between regenerative and preneoplastic nodules in cirrhotic liver.