Significance of telomerase activity in the diagnosis of small differentiated hepatocellular carcinoma

Abstract

Precise diagnosis of well-differentiated hepatocellular carcinoma (HCC) is sometimes difficult to establish. Telomerase activity was examined by telomeric-repeat-amplification protocol (TRAP) in 37 HCC nodules smaller than 3 cm in diameter, including 24 fine-needle-aspiration biopsy specimens, 22 non-tumor chronic-liver-disease tissues (9 chronic hepatitis and 13 liver cirrhosis) and 3 normal liver tissues. Telomerase activity was assayed by serially diluted samples and quantitated by using an internal telomerase assay standard (ITAS). Telomerase activity was detected in all HCC and in 11 of 22 non-tumor chronic-liver-disease tissues. Normal liver samples had undetectable telomerase activity. Cut-off level of telomerase activity for its practical usage in HCC diagnosis was tentatively set for 0.6 mg liver protein/ assay at 10-cell equivalent activity of a gastric-cancer cell line, MKN-1. This level was twice the highest activity in non-tumor chronic liver disease therefore, telomerase activity in all non-tumor liver samples was below this level. The telomerasepositive incidence exceeding this cut-off level was 73% (11/15) in well-differentiated HCC, 94% (16/17) in moderately differentiated HCC and 100% (5/5) in poorly differentiated HCC. Well-differentiated HCC showed low positivity by other diagnostic markers. 21% by AFP, 0% by PIVKA-II and 13% by angiography. The detection of telomerase activity may thus be a useful additional tool for precise and early diagnosis of small differentiated HCC, even when diagnosis is inconclusive by conventional techniques. Int.

Immuno-histochemical detection of human telomerase reverse transcriptase in human liver tissues

“…Since signal disappeared following pre-absorption of antibody with hTERT peptide used as immunogen, staining was speci®c for hTERT protein ( Figure 5b). As we have previously reported (Tahara et al, 1995;Nakashio et al, 1997), chronic liver diseases such as chronic hepatitis and liver cirrhosis exhibited low but signi®cant levels of telomerase activity. We did not know at the time of publication of those reports whether the weak telomerase activity was derived from telomerasepositive hepatocytes or from in®ltrating lymphocytes.…”

“…We have previously reported that HCCs exhibit a high incidence of telomerase activity, that non-cancerous liver tissues have feeble or no detectable activity, and that telomerase activity increases in accordance with degree of histological undi erentiation of HCC (Tahara et al, 1995;Nakashio et al, 1997). While the level of expression of hTERT mRNA and level of telomerase activity were not always well correlated in other tissues (Yasui et al, 1998Ouellette et al, 1999;Nakamura et al, 1999), they were well correlated in liver tissues, as we previously reported Takahashi et al, 2000).…”

“…Since these previous studies were based on the analyses in whole tissue extracts, it was unclear whether the level of telomerase activity observed re¯ected that in individual cells or the frequency in telomerase-positive cells. We also previously reported that such non-cancerous liver tissues as chronic hepatitis and liver cirrhosis exhibited low but signi®cant levels of telomerase activity (Tahara et al, 1995;Nakashio et al, 1997;Takaishi et al, 2000). We did not know whether the low telomerase activity in these tissues was derived from hepatic cells or migrating lymphocytes.…”

“…Thus, reactivation of telomerase represents an important step toward unlimited proliferation and inde®nite survival of cancer cells. We previously reported that hepatocellular carcinomas (HCC) exhibited a high incidence of telomerase activity and that telomerase activity increased in accordance with degree of histological undi erentiation of HCC (Tahara et al, 1995;Nakashio et al, 1997). The level of expression of hTERT mRNA determined by RT ± PCR was very well correlated, in both non-cancerous tissues and HCC tissues with telomerase activity level Takahashi et al, 2000).…”

“…The protein concentration of the extract was measured using a DC protein kit (Bio-Rad, Hercules, California, USA). Telomerase activity was assayed by the TRAP (telomeric repeat ampli®cation protocol) assay with some modi®cations (Kim et al, 1994;Tahara et al, 1995;Nakashio et al, 1997). In brief, an aliquot of extract containing 0.6 mg tissue protein was incubated with 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 63 mM KCl, 1 mM EGTA, 0.005% Tween-20, 50 mM dNTPs, 0.1 mg of TS primer, 5 attogram of Internal Telomerase Assay Standard (ITAS) and 2 U of AmpliTaq DNA polymerases at 208C for 30 min.…”