Telomerase activity as a predictive marker for recurrence of hepatocellular carcinoma after hepatectomy

The American Journal of Surgery 181 (2001) 284 –288

Telomerase activity as a predictive marker for recurrence of hepatocellular carcinoma after hepatectomy Takashi Kobayashi, M.D.*, Keiichi Kubota, M.D., Ph.D., Tadatoshi Takayama, M.D., Ph.D., Masatoshi Makuuchi, M.D., Ph.D. Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Manuscript received June 19, 2000; revised manuscript November 7, 2000

Abstract Background: Expression of telomerase activity and stabilization of telomeres are concomitant with attainment of immortality in tumor cells. Telomerase activity levels in hepatocellular carcinoma (HCC) may serve as a predictive marker for recurrence after surgery. Methods: Telomerase activity levels in HCC were measured in 37 patients undergoing hepatectomy by the telomeric repeat amplification protocol. The clinicopathologic factors and telomerase activity were analyzed to identify factors that were important in affecting recurrence of HCC and recurrence-free survival. Results: Telomerase activity was detected in 23 patients (62.2%), and was not significantly associated with seven other HCC-related factors. After a median follow-up of 34 months, 24 patients (64.9%) had recurrence of HCC. In univariate analyses, telomerase activity of more than 20 total product generated (TPG) and portal vein invasion were found to be significantly related to a shorter time to recurrence. Multivariate analysis demonstrated that telomerase activity (⬎20 TPG) was significantly related to an increased risk of recurrence (relative risk 2.36, 95% confidence interval 1.03 to 5.43, P ⫽ 0.04). Conclusions: Telomerase activity can be identified as an independent predictor for recurrence after resection of HCC. © 2001 Excerpta Medica, Inc. All rights reserved. Keywords: Hepatocellular carcinoma; Telomerase activity; Predictive marker for recurrence

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. Recent development of diagnostic modalities such as ultrasonography (US) and computed tomography (CT) have contributed to its earlier diagnosis [1], and sophisticated surgical techniques for liver resection have made hepatectomy a safe and effective treatment for HCC [1,2]. Nevertheless, recurrence of HCC still remains common even after curative surgery because of early intrahepatic spread through the portal venous system and a high incidence of associated liver cirrhosis responsible for multicentric hepatocarcinogenesis. Telomeres are specialized structures of chromosome ends that are composed of simple tandem repeats of

* Corresponding author. Tel.: ⫹81-3-3815-5411; fax: ⫹81-3-56843989. E-mail address: [email protected].

TTAGGG [3]. In normal somatic cells, the telomere gradually loses its sequence with each increasing replication until, eventually, a critical reduction of its length causes cell death due to chromosomal instability and incapability of replication [4]. In contrast to normal cells, telomere length of immortal cancer cell lines is maintained as the consequence of reactivation of telomerase, which is a ribonucleoprotein enzyme adding TTAGGG repeats onto telomere [5,6]. Thus, telomerase takes a lead role as a potential enzyme responsible for cell longevity. Recent studies have shown that telomerase activity is present in a wide variety of human tumors, including HCC [7–11]; however, few studies have examined the relationship between the postoperative prognosis of patients with HCC and telomerase activity in HCC [9,11]. This study was performed to investigate whether telomerase activity may serve as a helpful factor in predicting recurrence of HCC after resection.

0002-9610/01/$ – see front matter © 2001 Excerpta Medica, Inc. All rights reserved. PII: S 0 0 0 2 - 9 6 1 0 ( 0 1 ) 0 0 5 6 6 - 9

T. Kobayashi et al. / The American Journal of Surgery 181 (2001) 284 –288

Methods Thirty-seven consecutive patients who underwent curative resection for HCC from September 1996 to September 1997 were investigated prospectively. Among 37 patients (31 men and 6 women; median age 63 years, range 43 to 79), 11 had chronic hepatitis, 22 had liver cirrhosis, and 4 had liver fibrosis. Anatomical resection included hepatic lobectomy, segmentectomy, and subsegmentectomy; and nonatomical resection was defined as a limited resection of smaller part of the subsegment. After surgery, all patients were screened by US every 2 months and dynamic CT every 4 months. When recurrence was suspected, angiography was performed for diagnostic purposes. Recurrence was defined clinically as the appearance of a new lesion with radiologic features typical of HCC, as confirmed by two imaging methods [12]. Multicentric tumors were defined as tumors in the different segments after 3 or more years, if the initial tumor had had no cancer spread [13]. Samples of HCC and corresponding non-HCC liver tissue were obtained by surgical resection from the 37 patients and were immediately frozen in liquid nitrogen and stored at ⫺80°C until use. Informed consent was obtained from each patient included in the study. Telomerase activity was quantified using a TRAP-eze telomerase detection kit (Intergen, New York, NY), and was detected using a fluorescencebased TRAP method according to the manufacturer’s instruction [14]. Briefly, 5 mg of frozen samples were homogenized in 100 ␮L of ice-cold CHAPS Lysis Buffer (TRAP-eze) and incubated for 30 minutes on ice. After incubation, the lysates were centrifuged at 12,000 g for 20 minutes at 4°C. The supernatants were rapidly frozen and stored at ⫺80°C. The protein concentration was determined by Coomassie protein assay reagent (Pierce, Rockford, Illinois), and aliquots of extract containing 1 ␮g of protein were used for each TRAP assay. Extracts were incubated with 0.1 ng of cy-5-labeled TS primer: 5⬘-AATCCGTCGAGCAGAGTT-3⬘ in a master mix (TRAP-eze). After 30 minutes of incubation at 30°C, polymerase chain reaction was performed at 94°C/30 sec and 72°C/45 sec for 30 cycles. TSR8 (TRAP-eze) was used as an external positive control. The products were diluted with an equal volume of formamide dye solution, heated at 94°C for 5 minutes, and applied (5 ␮L/lane) to 10% denaturing gel containing 6 M urea fitted to an automated DNA sequencer (ALF red express DNA sequencer). During electrophoresis at 45 watts, the temperature of the gel was kept at 50°C. Data from the ALF red express DNA sequencer were collected and analyzed automatically by Allele Links software (American Pharmacia Biotech, Uppsala, Sweden). Each peak was quantified in terms of size, peak height, and peak area. Telomerase activity was quantified by the formula: TPG (total product generated) units/␮g protein ⫽ [(A/B)/(A in positive control/B in positive control)] ⫻ 100, where A is the total area of telomerase activity (50 bp, 56 bp, 62 bp, 68 bp, and so forth) and B is the area of internal control (36

285

bp). We measured the telomerase activity three times in every nodules of HCC as well as in corresponding non-HCC liver tissue, and present the mean values. The associations between telomerase activity level in HCC and individual variables, including the degree of tumor-cell differentiation according to Edmondson’s system [15], presence of capsule formation, portal vein invasion, intrahepatic metastasis, TNM clinical stage, tumor size, and serum alpha-fetoprotein (AFP) level, were analyzed using Spearman rank order and regression analysis. The following factors were analyzed with respect to their prognostic significance in recurrence: Child-Pugh classification, telomerase activity level in HCC, distance of tumor-free margin, capsule formation, portal vein invasion, intrahepatic metastasis, numbers of tumors, tumor size, serum AFP level, and hepatectomy. Recurrence-free survival rates were calculated by the Kaplan-Meier method and differences between recurrence-free survival curves were assessed with the generalized Wilcoxon test. The Cox proportional hazards model was used to identify factors most significantly related to recurrence-free survival. P values less than 0.05 were considered to indicate statistical significance.

Results Telomerase activity in HCC was detected in 23 of 37 specimens (62.2%). Telomerase activity levels were not significantly associated with tumor cell differentiation, capsule formation, portal vein invasion, intrahepatic metastasis, TNM clinical stage, tumor size, or serum alpha-fetoprotein level (Table 1). During a median follow-up of 34 months (range 28 to 44), HCC recurred in 24 of 37 patients (64.9%). The 1-year, 2-year, and 3-year recurrence-free survival rates were 56.8%, 51.4%, and 31.5%, respectively. When the cumulative recurrence rates were compared among various subgroups identified by the 10 predictive factors, telomerase activity in HCC (P ⫽ 0.03) and portal vein invasion (P ⫽ 0.03) were found to be significantly related to a shorter time to recurrence (Table 2). However, telomerase activity was identified as the only significant prognostic variable by multivariate analysis (relative risk 2.36, 95% confidence interval 1.03 to 5.43, P ⫽ 0.04; Table 3). In non-HCC liver tissues, telomerase activities were undetectable in all 13 patients who had no recurrence, whereas they were elevated in 7 of 24 patients who had recurrence (median 13.98 TPG, range 12.64 to 50.33 TPG).

Comments Telomerase activity was detected in 23 of 37 HCC specimens (62.2%). Previous series have reported widely disparate telomerase positive rates for HCC, ranging from 61.5% to 100% [7,9,11,16]. In the present study, telomerase

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Table 1 Association between telomerase activity in hepatocellular carcinoma and clinicopathologic data Number of patients Edmondson’s grading Grade I Grade II Grade III Capsule formation Absent Present Portal vein invasion Negative Positive Intrahepatic metastasis Negative Positive TNM clinical stage I II III IV Tumor size (mm) ⬉20 20–50 ⬎50 Alpha-fetoprotein level (ng/mL) ⬉100 ⬎100

Telomerase activity (TPG)

P value

0 (n ⫽ 14)

0–20 (n ⫽ 4)

20 ⬍(n ⫽ 19)

12 23 2

2 11 1

2 2 0

8 10 1

0.14

10 27

2 12

2 2

6 13

0.32

22 15

9 5

2 2

11 8

0.74

30 7

12 2

2 2

16 3

0.97

4 21 7 5

1 10 1 2

1 1 1 1

2 10 5 2

0.68

10 14 13

1 7 6

2 0 2

7 7 5

0.11

19 18

7 7

2 2

10 9

0.88

TPG ⫽ total product generated.

activity was observed in all stages of HCC, supporting the hypothesis that telomerase reactivation may be an early event in the malignant progression of HCC [17], whereas it is known to occur at a later stage in other carcinomas [18]. The correlation among telomerase activity in tumors, histopathologic data, and clinical data has been a matter of controversy [8 –11,16,19 –21]. In our study, the levels of telomerase activity in HCC were not significantly associated with the selected factors related to tumor status. Thus, telomerase activity in HCC may have a different oncologic significance than the histopathologic features of tumor. Patients with HCC may have microscopic lesions that cannot be removed by surgery. Therefore, we confirmed just after surgical interventions that patients were free from macroscopic evidence of tumor by intraoperative ultrasonography. Occult metastases of HCC present at the time of surgery probably became detectable during follow-up. We defined recurrence as the development of new lesions according to the predefined criteria [12,13], which have a diagnostic accuracy rate of 96% [22]. Since all new lesions in the present study were detected within 3 years after hepatectomy, we diagnosed clinically them as possibly true recurrences. HCC is well known to spread into the liver in the early stage via the portal venous system. Several prognostic variables, including portal vein invasion, have been reported as important risk factors for HCC recurrence after hepatec-

tomy [12,13,23]. In the present study, portal vein invasion and telomerase activity in HCC were significantly related to shorter time to recurrence. A higher incidence of microscopic portal invasion has been reported in patients with HCC who showed increased telomerase activity levels [9]. However, telomerase activity levels may not directly reflect invasiveness of tumor as portal vein invasion but rather the propensity to survive many selection steps in terms of the persistent proliferation required for tumor metastasis [24]. Indeed, our results indicated a lack of association between telomerase activity in HCC and the extent of tumor invasion to the portal vein. Tumors with more than certain levels of telomerase activity may acquire the ability to form metastatic foci far from the primary lesion and lead to recurrence through many steps. Telomerase activity in HCC can be measured preoperatively by needle biopsy [16], whereas microscopic portal invasion of tumor cells cannot be evaluated without surgical resection. In our study, telomerase activity in HCC was the only significant predictive variable for recurrence. Portal vein invasion, which was identified as being significantly associated with a shorter time until recurrence, had no independent prognostic significance because of the overriding effect of telomerase activity in HCC in multivariate analysis. In any event, measurement of telomerase activity in HCC could provide clinically useful information on the prognosis of patients with HCC after hepatectomy.

T. Kobayashi et al. / The American Journal of Surgery 181 (2001) 284 –288

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Table 2 Univariate analyses of prognostic factors for recurrence Factor

Number of patients

Overall Child-Pugh classification A B Telomerase activity (TPG) ⬉20 ⬎20 Tumor-free margin (mm) ⬉5 ⬎5 Capsule formation Absent Present Portal vein invasion Negative Positive Intrahepatic metastasis Negative Positive Number of tumors Simple Multiple Tumor size (mm) ⬉50 ⬎50 Alpha-fetoprotein level (ng/mL) ⬉100 ⬎100 Hepatectomy Nonanatomical resection Anatomical resection

Recurrence-free survival rates (%)

P value

1-year

2-year

3-year

37

56.8

51.4

31.5

28 9

64.3 33.3

57.1 33.3

29.5 33.3

0.36

18 19

77.8 36.8

66.7 36.8

50.0 13.2

0.03

25 12

44.0 83.3

40.0 75.0

28.8 41.7

0.17

10 27

80.0 48.1

60.0 48.1

50.0 25.0

0.10

22 15

72.7 33.3

68.2 26.7

34.1 26.7

0.03

30 7

63.3 28.6

56.7 28.6

32.0 28.6

0.23

26 11

65.4 36.4

61.5 27.3

36.9 18.2

0.14

24 13

62.5 46.2

58.3 38.5

33.3 30.8

0.16

19 18

57.9 55.6

57.9 44.4

42.1 25.0

0.54

8 29

50.0 58.6

37.5 55.2

25.0 31.0

0.42

The significance of the difference for each parameter was tested by the generalized Wilcoxon test. TPG ⫽ total product generated.

Recent studies have shown that several immortalized human cell lines restore their telomere length without detectable telomerase activity [25,26]. Thus, reactivation of telomerase may not always be necessary for some tumor cells to acquire immortality. Indeed, in our study, 14 of 37

Table 3 Multivariate analysis of prognostic factors for recurrence Factor*

Number of Chi Relative risk P patients square of recurrence† value‡

Telomerase activity (TPG) ⬉20 (0) 18 ⬎20 (1) 19

4.11

2.36 (1.03– 5.43)

patients (37.8%) showed the absence of telomerase activity in HCC. Another possible explanation for this absence is that degradation of essential telomerase templating RNA may have occurred during freezing and preparing of samples, as telomerase is a ribonucleoprotein [5,6]. At present, we cannot distinguish between these possibilities. Thus, the biologic significance of telomerase activity in HCC remains uncertain, and further studies on this matter will be needed. The present study suggests that the telomerase activity levels in HCC are of significant value in predicting the recurrence of HCC after hepatectomy.

0.04

* Forward and backward stepwise analyses were used to select this variable. † Relative risks were expressed as the exponential coefficients and reflected increased hazards of the 0-coded factor. Values in parentheses are 95% confidence intervals. ‡ P value was from Cox proportional survival analysis. TPG ⫽ total product generated.

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