Int. J. Cancer (Pred. Oncol.): 84, 529–532 (1999) r 1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer TELOMERASE ACTIVITY CORRELATES WITH HISTO-PATHOLOGICAL FACTORS IN UTERINE ENDOMETRIAL CARCINOMA Yasuhiko EBINA1*, Hideto YAMADA1, Takafumi FUJINO1, Itsuko FURUTA1, Noriaki SAKURAGI1, Ritsu YAMAMOTO1, Motonobu KATOH2, Mitsuo OSHIMURA2 and Seiichiro FUJIMOTO1 1Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, Sapporo, Japan 2Department of Molecular and Cell Genetics, Tottori University Faculty of Medicine, School of Life Science, Yonago, Japan Telomerase activity has been implicated in the progression of various human tumors. Our aim was to evaluate telomerase activity and to compare it with histo-pathological factors in uterine endometrial carcinoma, to look for possible correlations. Telomerase activity was measured by dilution analysis using a PCR-based telomeric repeat amplification method and detected in 31 of 35 primary endometrial carcinoma tumor specimens. High telomerase activity, detected after 100-fold dilution of extracts, was identified in 15 specimens. There was no significant correlation between the positive telomerase activity and tumor surgical stage or histopathological factors. However, high telomerase activity was significantly correlated with advanced surgical stage and with pelvic lymph node metastasis. Our findings suggest that an increase in telomerase activity may be associated with tumor progression and that its level may have a prognostic value in endometrial carcinoma. Int. J. Cancer (Pred. Oncol.) 84:529– 532, 1999. r 1999 Wiley-Liss, Inc. Human telomeres consist of 4 to 15 kb of hexameric DNA repeats (TTAGGG) located at the ends of chromosomes. Due to the inability of DNA polymerase to replicate the ends of doublestranded DNA, telomeres progressively shorten with each round of cell division. Telomeric ends are considered to play an important role in the protection of the chromosomal ends against aberrant recombination and end fusion. The progressive shortening of telomeres eventually results in chromosomal instability, leading to cellular senescence. Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends using a segment of its RNA component as a template. The development of a highly sensitive PCR-based assay for telomerase activity has made it possible to study a wide variety of tumors and normal tissues (Kim et al., 1994). Using this method, telomerase activity has been detected in 85% of gastric carcinomas (Hiyama E., et al., 1995), 85% of hepatocellular carcinomas (Tahara et al., 1995) and 93% of breast carcinomas (Hiyama et al., 1996). In gynecological malignancies, telomerase activity has been detected in 83% to 100% of uterine cervical carcinomas (Kyo et al., 1996; Garham et al., 1997), 82% to 100% of uterine endometrial carcinomas (Kyo et al., 1996, 1997; Brien et al., 1997; Garham et al., 1997; Saito et al., 1997; Shroyer et al., 1997; Yokoyama et al., 1998) and 86% to 96% of ovarian carcinomas (Kyo et al., 1996; Garham et al., 1997; Yokoyama et al., 1998). Correlations between telomerase activity and clinico-pathological factors such as tumor stage and grade in gastric and breast cancer have also been described (Hiyama E., et al., 1995, 1996). Use of telomerase activity as a prognostic marker of clinical outcome was proposed because it has been related to the malignancy potential of the above-mentioned tumors. In endometrial carcinoma, however, telomerase activity has never been correlated with histo-pathological and prognostic factors (Brien et al., 1997; Kyo et al., 1997; Shroyer et al., 1997). In the present study, using dilution analysis and the PCR-based telomeric repeat amplification method, we have analyzed telomerase activity in endometrial carcinoma tissues and looked for correlations between telomerase activity and histo-pathological factors. MATERIAL AND METHODS Tissue specimens Endometrial carcinoma tissues taken from primary tumor sites were obtained from 35 Japanese patients (age range 23 to 77 years) at the time of surgery. Eleven patients (31.4%) were pre-menopausal and 24 (68.6%) were post-menopausal. These patients had never been treated with irradiation, chemotherapy or hormonal therapy before tissue sampling. Specimens derived from primary lesions of endometrial carcinomas were frozen immediately after sampling and stored once at ⫺80°C until analysis. The presence of malignant cells in the sampled tissues was confirmed before analysis by microscopy, using cryostat sections. Histo-pathological data, including histological subtype, histological and nuclear grade, myometrium and cervical invasion, adnexal metastasis, pelvic lymph node and para-aortic lymph node metastasis, lymph-vascular space invasion and extra-uterine disease, were collected and compared with telomerase activity. Histological grade was determined as follows: well-differentiated (G1), moderately differentiated (G2) or poorly differentiated (G3) according to FIGO criteria. Lymph-vascular space invasion was considered present when tumor cells were within or attached to the wall of a vascular or lymphatic space lined by flattened endothelial cells. Cervical invasion was diagnosed when tumor cells were within or adhered to endocervical tissue. Telomerase assay Telomeric repeat amplification protocol (TRAP) assay was performed according to the methods described by Kim et al. (1994), using an internal telomerase activity standard (ITAS), to exclude the possibility of Taq DNA polymerase inhibitory activity in the tumor extracts. Each tissue specimen (approx. 100 mg, frozen at ⫺80°C) was washed in ice-cold buffer [10 mM HEPESKOH (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT], pelleted, then sufficiently homogenized in 200 ml ice-cold lysis buffer [1 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 60% glycerol, 0.5% CHAPS, 5 mM 2-mercaptoethanol, 0.1 mM AEBSF]. After 30-min incubation on ice and centrifugation at 16,000 g for 20 min at 4°C, the supernatant was collected and stored at ⫺80°C. The protein concentration in the extract was measured using a BCA protein assay kit (Pierce, Rockford, IL). A portion of the supernatant was included in each TRAP assay tube containing 50 µl of reaction mixture composed of 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, 50 µM dNTPs, 0.1 µg TS primer, 1 µg T4 gene 32 protein (BoehringerMannheim, Mannheim, Germany), 2 U Taq DNA polymerase (Takara Shuzo, Kyoto, Japan) and 0.1 µg CX primer at the tube bottom sequestered by Ampliwax (Perkin Elmer, Norwalk, CT). Telomeric repeat was added to TS primer and maintained at 23°C for 30 min. The assay tube was then processed through 31 temperature cycles at 95°C for 30 sec, 50°C for 30 sec and 72°C for *Correspondence to: Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, N15 W7, Kita-Ku, Sapporo 060, Japan. Fax: 81-11-706-7711. Received 31 March 1999 EBINA ET AL. 530 FIGURE 1 – Telomerase activity in endometrial carcinoma. Each extract prepared from tumor tissue was subjected to serial dilutions and analyzed by TRAP assay at 6 µg protein (standard condition), at 0.6 µg (10-fold) and at 0.06 µg (100-fold). Lysis buffer was used as a negative control and a uterine cervical cancer cell line (Siha) as a positive control for telomerase signals from 10, 100 and 1,000 cells. The ITAS band is an internal control for PCR amplification. Case C, which remained positive after 100-fold dilution, was classified as high. Case A, which was positive after 10-fold dilution and became negative after 100-fold dilution, was classified as low. Case B showed no telomerase activity. 90 sec. The final products were visualized by non-denaturing 10% PAGE and visualized by SYBR green I nucleic acid gel stain (FMC, Rockland, ME). The sensitivity of the non-RI assay was as high as that of the conventional RI-TRAP assay. We classified a sample as telomerase-positive when a 6-bp DNA ladder was observed and its intensity was equivalent to or stronger than that obtained using 10 cells of a uterine cervical cancer cell line (SiHa). Because there was variation in the intensity of the TRAP signals in the tumor specimens, we estimated telomerase activity using serially diluted extracts containing 6 µg, 0.6 µg (10-fold) and 0.06 µg (100-fold) protein. Telomerase activity detected after 100-fold dilution was classified as high, whereas that which disappeared in diluted extracts was classified as low (Fig. 1). Statistical analysis Statistical analysis was performed using Fisher’s exact test; p ⬍ 0.05 was considered to be statistically significant. RESULTS Telomerase activity was detected in 31 (88.6%) of 35 endometrial carcinoma tissues. Extracts from all telomerase-negative samples gave a positive signal with the internal telomerase assay standard, excluding the possibility of Taq polymerase inhibition. After semi-quantification of telomerase activity by serial dilution of each extract, the 31 tumors showing positive telomerase signals were divided into 2 groups: 15 tumors had high telomerase activity (i.e., retained a TRAP signal after 100-fold dilution of the extract) and 16 had low telomerase activity (i.e., expressed TRAP signals before dilution or after 10-fold dilution but not after 100-fold dilution of the extract). The incidence of positive telomerase activity was not statistically significant in regard to surgical stage. However, the incidence of high telomerase activity was significantly high in stage III or IV tumors (62.5%, 10 of 16) when compared with that in stage I or II tumors (26.3%, 5 of 19; p ⬍ 0.05; Table I). TABLE I – TELOMERASE ACTIVITY AND SURGICAL STAGE IN ENDOMETRIAL CARCINOMA Surgical stage Number I II III IV I ⫹ II III ⫹ IV Total 16 3 12 4 19 16 35 Telomerase activity Negative Low1 High2 3 1 0 0 4 0 4 9 1 5 1 10 6 16 4 1 7 3 5 10 15 1Positive using 6 or 0.6 µg of extract but negative using 0.06 µg of extract.–2Positive using 6, 0.6 or 0.06 µg of extract. The correlation between histo-pathological factors and telomerase activity is shown in Table II. There was no significant correlation between the frequency of positive telomerase activity and histological subtype, histological grade, nuclear grade, myometrial invasion, cervical invasion, adnexal metastasis, pelvic node metastasis, para-aortic node metastasis, lymph-vascular space invasion or extra-uterine disease. Tumor lesions from all 4 telomerase-negative patients were strictly confined to the uterus. All specimens with adnexal, pelvic and para-aortic lymph node metastases showed positive telomerase activity. The incidence of high telomerase activity was significantly high in patients with pelvic lymph node metastasis ( p ⬍ 0.05). Although all 7 specimens of extra-uterine disease had positive telomerase activity and tended to have high telomerase activity, this was not significant ( p ⫽ 0.10). When only post-menopausal patients were included in the statistical analysis, 4 of 15 (26.7%) tumors from stage I or II patients exhibited high telomerase activity and 7 of 9 (77.8%) tumors from stage III or IV patients showed high telomerase activity with statistical significance ( p ⬍ 0.05). Pelvic lymph node metastasis was also significantly correlated with high telomerase activity (presence of metastasis 66.7% vs. absence of metastasis 38.9%, p ⬍ 0.05). Thus, high telomerase activity significantly TELOMERASE ACTIVITY IN ENDOMETRIAL CANCER TABLE II – CORRELATION BETWEEN HISTO-PATHOLOGICAL FACTORS AND TELOMERASE ACTIVITY IN ENDOMETRIAL CARCINOMA Histo-pathological factors Histological subtype Endometrioid carcinoma Serous adenocarcinoma Histological grade G1 G2 G3 Nuclear grade 1 2 3 Myometrial invasion Tumor limited to endometrium Invasion to ⬍1/2 myometrium Invasion ⬎1/2 myometrium Serosal invasion Cervical invasion Negative Positive Adnexal metastasis Negative Positive Pelvic node metastasis Negative Positive Para-aortic node metastasis Negative Positive Lymph-vascular space invasion Negative Positive Extra-uterine disease Negative Positive Number Telomerase activity Negative Low1 High2 33 2 4 0 15 1 14 1 19 11 5 1 3 0 9 6 1 9 2 4 16 14 5 1 3 0 8 7 1 7 4 4 6 15 9 5 1 3 0 0 4 5 5 2 1 7 4 3 28 7 3 1 14 2 11 4 29 6 4 0 14 2 11 4 26 6 4 0 14 1 8 5 28 3 4 0 14 1 10 2 21 14 3 1 10 6 8 7 28 7 4 0 14 2 10 5 1Positive using 6 or 0.6 µg of extract but negative using 0.06 µg of extract.–2Positive using 6, 0.6 or 0.06 µg of extract. correlated with pelvic lymph node metastasis and advanced surgical stage, even in post-menopausal patients. DISCUSSION Uterine endometrial carcinoma is one of the common invasive malignancies of the female genital tract, and its prevalence is steadily increasing in Japan as well as in Western countries. However, information regarding the molecular mechanisms of endometrial carcinogenesis remains limited. Mutations of ras (Enomoto et al., 1990; Ignar-Trawbridge et al., 1992) and p53 (Okamoto et al., 1991; Risinger et al., 1992) genes are involved in the development and progression of endometrial carcinoma, but the incidence of mutation in these 2 genes is low, estimated at 10% to 46% and 13% to 14%, respectively (Enomoto et al., 1990; Okamoto et al., 1991; Ignar-Trawbridge et al., 1992; Risinger et 531 al., 1992). Our relatively new approach of the molecular investigation of telomere–telomerase interactions may provide additional insight into the mechanisms of endometrial carcinogenesis. Most normal human somatic cells exhibit undetectable levels of telomerase activity, whereas a majority of tumor cells express telomerase activity and shortened telomeres (Kim et al., 1994). In normal cells, telomerase is particularly expressed in embryonic cells (Wright et al., 1998), adult male germ-line cells (Wright et al., 1998), proliferative cells of renewable tissues such as basal cells of the epidermis (Harle-Bachor and Boukamp, 1996), circulating peripheral blood leukocytes (Hiyama K. et al., 1995) and normally cycling endometrium (Brien et al., 1997; Kyo et al., 1997; Saito et al., 1997). Kyo et al. (1997) have evaluated telomerase activity by semi-quantitative analysis and have shown that normal endometrium expresses telomerase activity, the level of which is regulated during the menstrual cycle. In contrast, atrophic endometrial samples from post-menopausal women exhibit no or only weak telomerase activity (Brien et al., 1997; Kyo et al., 1997; Saito et al., 1997; Shroyer et al., 1997), suggesting that residual telomerase activity in post-menopausal endometrium reflects loss of function in the basal endometrial lining, which is no longer capable of regeneration. Various investigations correlating telomerase activity with prognostic factors have yielded conflicting results. Studies of human gastric (Hiyama E., et al., 1995) and breast (Hiyama E., et al., 1996) cancer have shown that the frequency of lymph node metastasis in patients with positive telomerase activity in primary tumors was higher than that in patients without telomerase activity. In pancreatic carcinoma, the level of telomerase activity in primary tumors obtained surgically from patients with distant metastases is significantly higher than in primary tumors without metastases (Hiyama E., et al., 1997). In contrast, in hepatocellular (Tahara et al., 1995) and renal cell (Mehle et al., 1996) carcinoma, no relationship between telomerase activity and clinico-pathological parameters, such as histo-pathological grade, tumor size, DNA ploidy, stage and clinical outcome, has been noted. To our knowledge, in human endometrial carcinomas no correlation between telomerase activity and any histo-pathological prognostic factors has been observed (Brien et al., 1997; Shroyer et al., 1997). However, we have found here that high telomerase activity significantly correlates with the presence of pelvic lymph node metastasis and advanced surgical stage. These correlations were confirmed when only post-menopausal patients were analyzed. The presence of pelvic node metastasis and advanced surgical stage are indeed adverse factors that determine poor prognosis in patients with endometrial cancer. Endometrial carcinomas with high telomerase activity may be more aggressive than those without or with low telomerase activity. ACKNOWLEDGEMENTS We thank Drs. H. Hareyama (Sapporo City Hospital), S. Shimada (Kushiro Red Cross Hospital), M. Morikawa (Ohji General Hospital) and M. Oikawa (Hokkaido University) for the collection of samples. REFERENCES BRIEN, T.P., KALLAKURY, B.V., LOWRY, C.V., AMBROS, R.A., MURACA, P.J., MALFETANO, J.H. and ROSS, J.S., Telomerase activity in benign endometrium and endometrial carcinoma. Cancer Res., 57, 2760–2764 (1997). ENOMOTO, T., INOUE, M., PERANTONI, A.O., TERAKAWA, N., TANIZAWA, O. and RICE, J.M., K-ras activation in neoplasms of the human female reproductive tract. Cancer Res., 50, 6139–6145 (1990). carcinoma-derived skin keratinocytes. Proc. nat. Acad. Sci. (Wash.), 93, 6476–6481 (1996). HIYAMA, E., GOLLAHON, L., KATAOKA, T., KUROI, K., YOKOYAMA, T., GAZDAR, A.F., HIYAMA, K., PIATYSZEK, M.A. and SHAY, J.W., Telomerase activity in human breast tumors. J. nat. Cancer Inst., 88, 116–122 (1996). GARHAM, H., YOSHIDA, K., SUGINO, T., MARSH, G., MANEK, S., CHARNOCK, M., TARIN, D. and GOODISON, S., Telomerase activity in human gynecological malignancies. J. clin. Pathol., 50, 501–504 (1997). HIYAMA, E., KODAMA, T., SHINBARA, K., IWAO, T., ITOH, M., HIYAMA, K., SHAY, J.W., MATSUURA, Y. and YOKOYAMA, T., Telomerase activity is detected in pancreatic cancer but not in benign tumors. Cancer Res., 57, 326–331 (1997). HARLE-BACHOR, C. and BOUKAMP, P., Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and HIYAMA, E., YOKOYAMA, T., TATSUMOTO, N., HIYAMA, K., IMAMURA, Y., MURAKAMI, Y., KODAMA, T., PIATYSZEK, M.A., SHAY, J.W. and MATSUURA, 532 EBINA ET AL. Y., Telomerase activity in gastric cancer. Cancer Res., 55, 3258–3262 (1995). HIYAMA, K., HIRAI, Y., KYOIZUMI, S., AKIYAMA, M., HIYAMA, E., PIATYSZEK, M.A., SHAY, J.W., ISHIOKA, S. and YAMAKIDO, M., Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J. Immunol., 155, 3711–3715 (1995). IGNAR-TRAWBRIDGE, D., RISINGER, J.I., DENT, G.A., KOHLER, M., BERCHUCK, A., MCLACHLAN, J.A. and BOYD, J., Mutation of the Ki-ras oncogene in endometrial carcinoma. Amer. J. Obstet. Gynecol., 167, 227–232 (1992). KIM, N.M., PIATYSZEK, M.A., PROWSE, K.R., HARLEY, C.B., WEST, M.D., HO, P.L.C., COVIELLO, G.M., WRIGHT, W.E., WEINRICH, S.L. and SHAY, J.W., Specific association of human telomerase activity with immortal cells and cancer. Science, 266, 2011–2015 (1994). KYO, S., KANAYA, T., ISHIKAWA, H., UENO, H. and INOUE, M., Telomerase activity in gynecological tumors. Clin. Cancer Res., 2, 2023–2028 (1996). KYO, S., TAKAKURA, M., KOHAMA, T. and INOUE, M., Telomerase activity in human endometrium. Cancer Res., 57, 610–614 (1997). MEHLE, C., PIATYSZEK, M.A., LJUNGBERG, B., SHAY, J.W. and ROOS, G., Telomerase activity in human renal cell carcinoma. Oncogene, 13, 161–166 (1996). OKAMOTO, A., SAMESHIMA, Y., YAMADA, Y., TESHIMA, S., TERASHIMA, Y., TERADA, M. and YOKOTA, J., Allelic loss on chromosome 17p and p53 mutations in human endometrial carcinoma of the uterus. Cancer Res., 51, 5632–6536 (1991). RISINGER, J.I., DENT, G.A., IGNAR-TROWBRIDGE, D., MCLACHLAN, J.A., TSAO, M.S., SENTERMAN, M. and BOYD, J., p53 gene mutations in human endometrial carcinoma. Mol. Carcinogenesis, 5, 250–253 (1992). SAITO, SCHEL, T., SCHNEIDER, A., MARTEL, N., MIZUMOTO, H., BULGAY-MAERM., KUDO, R. and NAKAZAWA, H., Proliferation-associated regulation of telomerase activity in human endometrium and its potential implication in early cancer diagnosis. Biochem. biophys. Res. Comm., 231, 610–614 (1997). SHROYER, K.R., STEPHENS, J.K., SILVERBERG, S.G., MARKHAM, N., SHROYER, A.L., WILSON, M.L. and ENOMOTO, T., Telomerase expression in normal endometrium, endometrial hyperplasia, and endometrial adenocarcinoma. Int. J. Gynecol. Pathol., 16, 225–232 (1997). TAHARA, H., NAKANISHI, T., KITAMOTO, M., NAKASHIO, R., SHAY, J.W., TAHARA, E., KAJIYAMA, G. and IDE, T., Telomerase activity in human liver tissues: comparison between chronic liver disease and hepatocellular carcinomas. Cancer Res., 55, 2734–2736 (1995). WRIGHT, W.A., PIATYSZEK, M.A., RAINEY, W.E., BYRD, W. and SHAY, A.W., Telomerase activity in human germline and embryonic tissues and cells. Develop. Genet., 18, 173–179 (1998). YOKOYAMA, Y., TAKAHASHI, Y., SHINOHARA, A., LIAN, Z. and TAMAYA, T., Telomerase activity in the female reproductive tract and neoplasms. Gynecol. Oncol., 68, 145–149 (1998).