232 Telomerase as a Tool for the Differential Diagnosis of Human Hepatocellular Carcinoma Kazuhiro Nouso, M.D. Yoshiaki Urabe, M.D. Toshihiro Higashi, M.D. Harushige Nakatsukasa, M.D. Naoki Hino, M.D. Kouzou Ashida, M.D. Nobuyuki Kinugasa, M.D. Keigo Yoshida, M.D. Shuji Uematsu, M.D. Takao Tsuji, M.D. First Department of Internal Medicine, Okayama University Medical School, Okayama, Japan. BACKGROUND. Telomerase activation is thought to be essential for the immortality of cancer cells. We measured telomerase activity in human liver samples, including hepatocellular carcinoma (HCC),and evaluated this assay as a tool for the diagnosis of HCC using 21-gauge (Zl-G)-needlebiopsy specimens. METHODS. Ninety-four liver samples (27 HCC, 27 liver cirrhosis, 37 chronic hepatitis, and 3 normal liver) that were surgically resected or biopsied with a 12-gauge Silverman needle and 13 HCC samples that were biopsied with a 21-G needle were analyzed for telomerase activation. RESULTS. Eleven of 29 (38%) tumor-bearing liver samples were weakly telomerasepositive, whereas telomerase activity was observed infrequently in nontumor-bearing liver samples (G of 35; 17%) and in normal liver samples (0 of 3: 0%). The positivity of surgical samples for well differentiated, moderately differentiated, and poorly differentiated HCC was 88% (7 of 8), 87% (13 of 151, and 0% (0 of 21, respectively. In telonierase-positive HCC, 43% (3 of 7) of well differentiated samples were weakly positive, whereas 92% (12 of 13) of moderately differentiated samples were strongly positive. The difference in the tumor sizes and viral marker status did not affect the activity. The telomerase activity of the 21-G-needle biopsied specimens showed no significant difference from that of the surgical samples. The positive incidence of 21-G specimens was 80% (8 of 10) and 100% (2 of 2) in well differentiated HCC and moderately differentiated HCC, respectively. CONCLUSIONS. An incremental positivity of telomerase was observed during hepatocarcinogenesis. The use of this assay in 21-G-needle biopsy specimens may be useful in clinical examination. Cancer 1996; 78232-6. 0 1996 American Cancer Society. KEYWORDS: hepatocellular carcinoma, telomerase, telomere, needle biopsy. This work was supported by a grant in aid for cancer research from the Ministry of Health and Welfare of Jaoan. The authors thank Dr. Yoshiyuki Shimamura (Chiba-Nishi Hospital, Japan) and Or. Junnosuke Shimamura (Kurashiki Central Hospital) for providing a portion of the human liver samples. Address for reprints: Kazuhiro Nouso, M.D., The First Department of Internal Medicine, Okayama University Medical School, 2-5-1 Shikatd-cho, Okayama-city, Okayama 700 Japan. Received December 14, 1995; revision received March 29. 1995; accepted March 29, 1996. 0 1996 American Cancer Society T he telomere is a structure of chromosome ends that is comprised of simple tandem repeat sequences.' In humans, 10-15 kilobases of TTAGGG repeats are found at the ends of all chromosomes, and the integrity of this region protects termini from the devastating attack of exonuclease and liga~e.'.~ In normal somatic cells, telomeric DNA gradually loses its sequence with successive repeats of replication. After a certain number of doublings, the cells eventually stop dividing at the senescence stage known as M1. Thus, telomere reduction is thought to be a biologic clock regulating the life span of a cell."' In contrast to normal cells, the telomere length is known to be maintained in immortal cancer cell lines. This telomere length preservation has been revealed to be the consequence of telomerase activat i ~ n . Ribonucleoproteins ~-~ known as telomerases synthesize G-rich telomeric repeats using their complementary RNA sequence as a template.' The activity has also been detected in human cancers in addition to many immortal cell lines and cancer cell lines, but not in Telomerase in HCClNouso et al. 233 normal cell lines or tissue^.^.'"^^^ The immortality of the cancer cells is a key aspect of tumor growth and metastasis because it affords the cell freedom from aging, allowing the tumor to expand indefinitely. Hepatocellular carcinoma (HCC) develops mainly in liver cirrhosis (LC),which consists of numerous regenerative nodules. The development of imaging modalities such as helical computed tomography'5 and ultrasonography now afford the early detection of very small nodular lesions whose diameters are less than 1 cm. However, it is very difficult to differentiate small HCC from large hyperplastic nodules or dysplastic nodules even on histologic examination because of the limited sample volume. Furthlermore, it is still uncertain whether all of these nodules have a potential for malignancy. Therefore, it is important to ascertain the immortality of these cells for differential diagnosis and for assessment of the grade of malignancy. To analyze the telomerase activity of small samples and to expand the applications of this assay in clinical use, we examined whether 21 -gauge (21-GI-needle biopsied specimens could be subjected to this assay because percutaneous ethanol injection thel-apyIGis now widely used in Japan, whereas surgical resection of small hepatic tumors is rarely performed. In this study we analyzed the telomerase activity of many human hepatic specimens with particular emphasis on whether the 21-G-needle biopsied specimens are appropriate for this assay. MATERIALS AND METHODS Ninety-four liver samples ( 2 i HCC, 27 LC, 37 chronic hepatitis, and 3 normal liver) that were surgically resected or biopsied with a 12-gauge Silverman needle and 13 HCC samples that were biopsied with 21-G needle were analyzed for telomerases. All nontumorbearing samples were obtained by liver biopsy from patients before interferon therapy. We confirmed that they were tumor free by careful examination of ultrasonography and computed tomography. Of the 84 patients, 28 (33%) were women; patients' ages ranged from 15 to 80 years (mean, !54.9 years). Nineteen patients (23%)had hepatitis B viirus and 51 patients (61%) were positive for the hepatitis C virus. Informed consent was obtained from all patients for the experimental use of the samples. The samples were immediately frozen with dry ice after biopsy or surgery and stored at -80 "C. Telomerase activity was analyzed according to the method previously described with some modification." The samples (20-60 mg) were minced with scissors in 1 mL of wash buffer (10 mM Hepes, 1.5 mM MgCI,, 10 mM KCl, and 1 mIvl dithiothreitol) then ho- FIGURE 1. Autoradiogram of telomerase assay. Tissue extracts (6 pg) were used for this assay. Note that the cirrhotic portion in Patient 2 was telornerase positive. CH: chronic hepatitis; LC. liver cirrhosis: HCC: hepatocellular carcinoma. mogenized with 200 pL of lysis buffer (10 mM TrisHCI IpH 7.51, 1 mM MgCl, , 1 mM ethylene glycol-bis (P-aminoethyl ether) N,N,N',N'-tetraacetic acid, 0.1 M phenylmethylsulfonyl fluoride, 5 mM P-mercaptoethanol, 0.5% 3-[ (3-~holamidopropyl)-dimethylammoniol- 1-propanesulfate, and 10% glycerol). For the extraction of 21 -G-needle biopsied samples, 200 pL, of the wash buffer and 20 pL of the lysis buffer were used. After incubation for 25 minutes, the lysate was centrifuged at 15,000 g for 20 minutes. The supernatant fluid was stored at -80 "C. All procedures were performed at 4 "C. The extracted samples (6 p g or 0.6 pg of protein) were used for semiquantitative telomerase Fifty pL of the assay mixture containing 20 mM Tris-HCI (pH 8.31, 1.5 mM MgCL, 63 mM KCl, 0.005% Tween-20, 1 mM ethyleneglycoltetraacetic acid (EGTA), 50 pM deoxynucleoside triphosphate, 0.2 pL of [(I- "Pldeoxycytidine triphosphate (dCTP) (Amersham Japan, Tokyo, Japan), 0.1 pg of TS 234 CANCER July 15,1996 / Volume 78 / Number 2 TABLE 1 Telomerase Activity in Surgical Specimens of HCC Tissues Differentiation Well Moderate Poor Total Size s 2 cm 2< 5 5 cm >5 cm Total Viral markers HBsAg t HCVAb t Nan B Non C tt t - Posltive/no. of samples 4 12 0 16 3 1 0 4 1 2 2 5 718 (87.5%) 13/15 (86.7%)INS 0/2 (0%) 20125 (80%) 5 9 2 16 0 3 0 2 3 5 5/5 (100%) 12/14 (85.7%) NS 3 8 5 0 3 1 3 1 3/6 (50%) 11/12 (91.7%) NS 1 617 (85.7%) 1 4 3 / 6 (50%) 20/25 (80%) I I HCC: hepatocellular carcinoma: t t: strongly positive telomerase activity: t: weakly positive telomerase activity; -: negative telomerase activity;HBsAg: hepatitis B surface antigen; HCVAb hepatitis C virus antibody; NS: not significant [P> 0.05). TABLE 2 Telomerase Activity in Non-HCC Tissues With HCC Chronic hepatitis Liver cirrhosis Total Without HCC Chronic hepatitis Liver cirrhosis Total Normal liver tt t - Positivelno. of samples 0 0 3 8 11 7 11 18 8119 (42.1%) 11/29 (37.9%)a 0 0 0 0 5 1 6 0 22 0 7 29 3 1 5/27 (18.5%) NS 118 (12.5%) 6/35 (17.1%)a 013 (0%) HCC hepatocellular carcinoma; t t:strongly positive telomerase activity t:weakly positive telomerase activity; -: negative telomerase activity;NS: not significant (P > 0.05). ".05 c: P < 0.1. oligonucleotide, 1 pg of T4g32 protein (Boehringer Mannheim, Mannheim, Germany), bovine serum albumin (0.1 mg/mL, Sigma, St. Louis, MO), 2 U of Taq DNA polymerase (Perkin Elmer Applied Biosystems Japan, Chiba, Japan), and 1 p L of extracted samples were placed into the assay tube sealed with 0.1 pg of CX primer with Ampli Wax (Perkin Elmer). After incubation at 24 "C for 30 minutes, the tubes were heated at 90 "C for 90 seconds and subjected to 31 polymerase chain reaction cycles at 94 "C for 30 seconds, 52 "C for 30 seconds and 72 "C for 45 seconds. Six pL of the reactant were analyzed by electrophoresis in 0.5 x Tris-borate ethylenediamine tetraacetic acid (EDTA) on 15% polyacrylamide nondenaturing gel. We confirmed the activity by treating the samples with RNAse. All pro- cedures were performed in duplicate and the reproducibility was confirmed. Samples that were positive at 0.6 bg (24-hour exposure), positive at 6 pg (48-hour exposure), and negative at 6 pg (48-hour exposure) were denoted as strongly positive, weakly positive, and negative, respectively. The degree of cell differentiation was classified according to the Japanese general rules for primary liver cancer." Statistical significance was evaluated by means of the chi-square test. RESULTS The telomerase activity of HCC obtained as a surgical sample was observed in 20 of 25 samples (Fig. 1, Table 1).Positivity was observed in 88% (7 of 8) of well differentiated HCC and 87% (13 of 15) of moderately differ- Telomerase in HCC/Nouso et al. 235 (P < 0.05) than that of well differentiated HCC (9 of 18). DISCUSSION FIGURE 2. Telomerase assay using hepatocellular carcinoma samples of different weights. A: undiluted; B: diluted 10 times; C: treated with RNAse. The numbers at the top of the figure represent the weights of the hepatocellular carcinoma samples used for each extraction. entiated HCC. Although no significant difference in the incidence was observed between the stages of differentiation, 43% (3 of 7) of well differentiated HCC were weakly positive whereas 9;!% (12 of 131 of moderately differentiated HCC were strongly positive. Larger tumors tended to show a lower incidence of positivity; however, no significant difference was observed. The viral marker status did not affect the activity. The activity in specimens from chronically diseased livers and normal livers is shown in Table 2. Eleven of 29 samples of tumor-bearing livers (38%) were weakly positive. This incidence tended to be higher than that in nontumor-bearing livers (6 of 35; 17%) and that in normal livers (0 of 3; 0%). None of the samples showed strongly positive telomerase activity. In the sensitivity analysis of this assay, the same band intensities were obtained in the range from 2 mg to 10 mg (Fig. 2). This might be sensitive enough to analyze 2 1-G-needle biopsied specimens because the weight of a 6-mm length of a needle-biopsied specimen is approximately 3 mg (data not shown). The data for the telomerase activity of the 21-G-needle biopsied specimens showed a pattern very similar to that of the surgical samples, and no significant difference was observed between these two groups (Table 3). The positivity incidence of 21-G specimens was 80% (8 of 10) and 100%(3 of 3) in well differentiated and moderately differentiated HCC, respectively. By collecting both biopsied and surgical samples together, the incidence of strongly positive telomerase activity in moderately differentiated HCC (15 of 18) was significantly higher Since the telomeric repeat amplification protocol” was developed, many human tissues have been analyzed for telomerase activity. Most of the reports indicate that the activitywas found to be specific to tumors with malignant potential, except germ cells and hematopoietic stem ~e1ls.l~ Our preliminary data also indicated that the liver cancer cell line PLC/PRF5 was telomerase active. No telomerase activity was observed in cases of familiar adenomatous polyp, which is a frequent prodrome of colon c a r ~ i n o m a .The ’ ~ precursor of HCC is clearly chronic liver diseases, especially LC. But unlike in colon carcinoma, the presence of large regenerative nodules and sometimes dysplastic nodules makes the diagnosis of HCC difficult. Even after the diagnosis of HCC, uncertainties still remain as to whether these tumors have a phenotype of cancer. The very low incidence of telomerase activation in LC, which is comprised of regenerative nodules, and the lower rate of strongly positive telomerase activity in well differentiated HCC than in moderately differentiated HCC indicate the progressive increase of telomerase activity according to the process of hepatocarcinogenesis. These observations also correlate with the invariant telomere length ratio (telomere length of the tumor/telomere length of the surrounding tissue) for moderately differentiated HCC, (ranging in size from 4 cm to 10 cm), which must be smaller in larger tumors in the absence of telomerases.’’ We observed no telomerase activity in poorly differentiated HCC and a tendency for larger tumors to show a lower incidence of telomerase activity. Poorly differentiated HCC and large HCC often present with necrosis; hence, this characteristic might affect the results. In addition, two poorly differentiated HCC grew very rapidly and transcatheter arterial embolization had to be performed before tumor biopsy. This procedure must have been related to necrosis formation. HCC frequently recurs after treatment. HCC-bearing livers are usually damaged by frequent and repeated cell necrosis and regeneration. The chronic liver injury makes it difficult for clinicians to resect or coagulate the HCC with ethanol with a large safety margin. This treatment limitation is associated with a high incidence of metastasis. The assay is sensitive enough to detect a single cell that has active telomerase. Although there is weak telomerase activity in some nontumor-bearing liver samples, this assay can be used as a tool for detecting micrometastasis and for making decisions regarding further treatment. The low incidence of telomerase activation in chronic 236 CANCER July 15, 1996 / Volume 78 / Number 2 TABLE 3 Telomerase Activitv in HCC Tissues Biopsied with a 21-Gauge Fine Needle Well Moderately tt t - PositivelNo. of samples 5 3 3 2 0 */lo(100%) (80%) NS 313 0 1 HCC: hepatocellular carcinoma; t t:strongly positive telonierase actiLity: t:weakly positive telomerase activity; -: negative telomerase activity; NS not significant (P> 0.05). hepatitis (CH) (17%)and LC without HCC and its frequent activation in HCC-bearing liver tissues (38%) support this conclusion. We have found the incidence of positivity of telomerase for CH and LC without HCC to be lower than that reported for telomerase-active nontumor-bearing CH and LC (greater than 50%).14In the process of optimizing our assay, we chose a higher annealing temperature (52 "C) than used by Tahara et al.l4 (50 "C) and this small modification might have affected the results. 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