A new predictive factor for hepatocellular carcinoma based on two-dimensional electrophoresis of genomic DNA

Abstract

Molecular genetic analyses have clari®ed that accumulation of genomic changes provides important steps in carcinogenesis and have identi®ed a number of valuable genetic markers for certain cancers. To date, however, no prognostic markers have been identi®ed for hepatocellular carcinoma (HCC). In this study, we used restriction landmark genomic scanning (RLGS), a new high-speed screening method for multiple genomic changes, to detect unknown genetic alterations in HCC. Thirty-one HCC samples and their normal counterparts were examined by RLGS. Eight spot changes were common in several cases, and all were seen only on the HCC pro®le. Five of these spots were detected in more than 12 of 31 cases (38.7%). Viral infection had no in¯uence on changes in the RLGS spots. The disease-free survival rate for patients with 516 changed RLGS spots was signi®cantly lower than that for patients with fewer changed RLGS spots (415 spots) (P50.001). In multivariate analysis, the number of changed spots was proven to retain an independent prognostic value (relative risk 1.095: P=0.0031). These results suggest that the number of changed RLGS spots may be a useful biological marker for recurrence of HCC.

Correlation of postoperative recurrence in hepatocellular carcinoma with demethylation of repetitive sequences

“…We have reported that the di erence between non-cancerous liver tissue and HCC in the number of altered RLGS spots was signi®cantly correlated with postoperative disease recurrence. This had been found to be a more useful marker for HCC prognosis than the conventional factors, as documented in our previous report (Itano et al, 2000). This is interpreted to imply that such markers re¯ect genomic changes more closely associated with the actual process of hepatocarcinogenesis than the changes detected by conventional methods.…”

“…Global DNA hypomethylation has been frequently observed in many types of cancers and is correlated with prognostic factors in several of these Kim et al (1994); Cravo et al (1996); Soares et al (1999) and Shen et al (1998) reported that decreased levels of global DNA methylation in HCC are correlated with tumor characteristics. In our previous paper we showed that the number of altered RLGS spots in HCC compared to non-cancerous liver tissue was signi®cantly correlated with postoperative recurrence of HCC (Itano et al, 2000), and here we show that the intensity of the HTRS and CNIC spots, in other words, the degrees of demethylation in HTRS and CNIC, were signi®cantly correlated with the number of altered RLGS spots (Figure 3a,b). The above considerations strongly suggest that the number of altered RLGS spots and demethylation in repetitive sequences might be correlated with global DNA hypomethylation.…”

“…As reported in a previous paper, the intensity levels of all 1214 spots in a RLGS pro®le of a hepatitis virusnegative non-cancerous liver tissue resected because of liver metastasis of colon cancer were quanti®ed and on this basis the spots fell into several discrete peaks. The minimum peak represented the haploid level (1500 ± 3000 pixel value6 inches 2 ) and the second peak, the intensity of which was two-fold higher than that of the minimum peak, represented the diploid level (4000 ± 5500 pixel value6inches 2 ) (Itano et al, 2000). This`diploid level' was then taken as the basic standard against which to estimate spot intensities in this study.…”

The study of aberrant methylation in cancer via restriction landmark genomic scanning

“…Thirteen out of 16 patients whose tumors showed 16 or greater changes in their RLGS profiles had recurrence, while only two out of 15 patients whose tumors showed 12 or less changes recurred. Furthermore, the number of RLGS profile changes was more predictive than the three previously used prognostic indicators (Itano et al, 2000).…”

“…However, it is not uncommon for recurrence to occur in patients who are negative for all three of these factors. After RLGS analysis was performed on 31 HCC tumors and corresponding normal tissues, correlations were studied between RLGS profile changes and post-operative recurrence (Itano et al, 2000). Post-operative recurrence correlated significantly with 16 or more RLGS spot changes including both hypo-and hypermethylation events.…”