Dedifferentiation of human hepatocellular carcinoma Up-regulates telomerase and Ki-67 expression

Hypothesis: Dedifferentiation of human hepatocellular carcinoma (HCC) may influence telomerase activation and Ki-67 expression. Design: Laboratory study using human HCC specimens. Setting: University hospital. Patients: Twelve patients with HCC with specific morphologic patterns (nodule-in-nodule [n=4] or confluent multinodular [n=8] type) and histological heterogeneity and who had undergone curative hepatectomy were studied. Of these, 8 patients had 2 different histological grades of HCC cells distributed at various nodules but within the same tumor; 3 patients, 3 different histological grades; and 1 patient, all 4 different histological grades. Intervention: Tissue samples were retrieved from each nodule of the tumor and not mixed with one another. A total of 42 cancerous tissues from different distinctive nodules of the 12 patients were taken for telomerase and Ki-67 study, and corresponding noncancerous counterparts (n=12) served as healthy control samples. Telomerase activity was assayed by the telomerase repeat amplification protocol. Expression of messenger RNA (mRNA) by the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) was determined using reverse transcription polymerase chain reaction. The relative telomerase activity and hTERT mRNA in each tissue sample was quantified using densitometry and expressed as a percentage of the standardized HeLa cell line. Immunostaining with anti-Ki-67 antibody was used to detect Ki-67 and was expressed as Ki-67 labeling index. Main Outcome Measures: Telomerase activity, hTERT mRNA, and Ki-67 labeling index stratified by different histological gradings in each patient was analyzed. The correlations between telomerase activity and hTERT mRNA and between telomerase activity and expression of Ki-67 were plotted. Results: Telomerase activity increased from more to less differentiated foci of HCC cells in each case (generalized linear model, P<.001). Mean ± SD expression of hTERT mRNA in 43 cancerous tissue samples, even those 4 with negative telomerase activity, was distinguishable from that of the noncancerous controls (0.84±0.23 vs 0.41±0.11; t test, P=.008). Telomerase activity was correlated to hTERT mRNA expression (Pearson correlation, r²=0.56; P<.001). The Ki-67 labeling index increased from more to less differentiated foci of HCC in each case (generalized linear model, P<.001). Expression of Ki-67 correlated with telomerase activity within differently graded areas within individual tumors (Pearson correlation, r²=0.38; P<.001). Conclusion: Using the model of human HCC with histological heterogeneity, we determined that dedifferentiation of human HCC induces telomerase activation and Ki-67 expression.