Telomerase Activity as an Indicator of Potentially Malignant Adrenal Tumors
Yasuhiro Hirano, M.D.1 Kimio Fujita, M.D.1 Kazuo Suzuki, M.D.1
Tomomi Ushiyama, M.D.1 Yoshihisa Ohtawara, Ph.D.1 Fumio Tsuda, Ph.D.2
1 Department of Urology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
2 Department of Medical Sciences, Toshiba Gen- eral Hospital, Tokyo, Japan.
BACKGROUND. Telomerase is an enzyme that adds repeated telomere sequences to the ends of chromosome arms. It helps maintain both the length of telomere and infinite cell proliferation. In recent years, telomerase activity has been considered an important characteristic that differentiates between normal and cancerous cells. Because the authors often encountered difficulties in distinguishing between benign and malignant adrenal tumors, they investigated whether the expression of telomerase activity could distinguish potentially malignant adrenal tumors.
METHODS. The authors examined telomerase activity in 48 samples of adrenal tumor tissue and 27 samples of adjacent normal adrenal tissue. All samples were obtained from 48 patients who underwent surgery at Hamamatsu University Hospital in Hamamatsu, Japan. Based on the clinical and postoperative pathologic examinations, 45 samples were diagnosed as benign and 3 were diagnosed as malignant. Telomerase activity was examined using a telomerase repeat amplifi- cation protocol (TRAP) assay.
RESULTS. Of the 48 adrenal tumor samples, 7 (14.6%) had telomerase activity. All adjacent normal adrenal tissues were negative for telomerase activity. Of the telomerase positive samples, two were clinically known adrenocortical carcinoma, and another was metastatic adrenal tumor from lung carcinoma. Four other telomerase positive samples were diagnosed as benign after clinical and initial pathologic examinations. However, two of the patients from whom these samples were taken developed metastatic lesions after adrenalectomy.
CONCLUSIONS. A telomerase assay of adrenal tumors may help predict their ma- lignant potential. Cancer 1998;83:772-6. @ 1998 American Cancer Society.
KEYWORDS: adrenal tumor, telomerase, telomere.
T elomere is the special structure of a chromosomal end, composed of long tandem repeats of TTAGGG in humans.1 It protects the terminal of the chromosome from the devastating attack of exonu- clease and ligase.1 In normal somatic cells, telomeres gradually shorten their sequences after repeats of replication.2 After a certain number of cell proliferations, telomeres become shortened, and then normal cells enter a stage of growth arrest.3 Therefore, the life span of a normal cell is limited, and its replicative capacity is correlated with the length of the telomeres. The action of telomere shortening in normal somatic cells is viewed as “a mitotic clock”.3-5
Telomerase is an enzyme that adds repeated telomere sequences to chromosomal ends and contributes to maintaining the length of telomere. It is detected in cancer cell lines in vitro and most immortal cells in vitro. In addition, it has recently been reported that telomer- ase is expressed in various human cancer tissues.6-16 Telomerase is inactive in adult somatic cells, except for testis cells and stem cells of
Presented in part at SIU Montreal 1997, Societe Internationale d’Urologie 24th World Congress, Montreal, Canada, September 11, 1997.
Address for reprints: Yasuhiro Hirano M.D., Depart- ment of Urology, Hamamatsu University of School of Medicine, 3600 Handa-cho, Hamamatsu, 431- 3192, Japan.
Received January 13, 1998; accepted February 2, 1998.
| Pathologic diagnosis | No. of telomerase positive samples/ total no. of samples (%) |
|---|---|
| Cortical tumor | |
| Functioning adenoma | 2/27 (7.4%) |
| Nonfunctioning adenoma | 0/5 (0%) |
| Cortical hyperplasia | 1/7 (14.3%) |
| Functioning cortical cancer | 1/1 (100%) |
| Nonfunctioning cortical cancer | 1/1 (100%) |
| Medullary tumor | |
| Pheochromocytoma | 1/6 (16.7%) |
| Metastasis from lung carcinoma | 1/1 (100%) |
| Total | 7/48 (14.6%) |
| Adjacent normal adrenal tissue | 0/27 (0%) |
| Total | 7/75 (9.3%) |
regenerative tissues.6,18-22 Therefore, it is important to detect the reactivation of telomerase.
We often encounter difficulties in distinguishing benign and malignant adrenal tumors. Histologically benign adrenal tumor sometimes recurs locally or ap- pears as a metastatic lesion after adrenalectomy. Te- lomerase activity was reported by Hiyama et al.1º in pediatric neuroblastoma that arose from the adrenal medulla. However, it has not been reported for an- other kinds of tumors of the adrenal gland, such as pheochromocytoma and adrenocortical tumors, in adults. In this study, we investigated whether the ex- pression of telomerase activity is observed and useful in distinguishing potentially malignant adrenal tu- mors, which are difficult to diagnose even pathologi- cally.
MATERIALS AND METHODS
We examined telomerase activity in 75 samples. All samples were obtained from 48 patients (ranging in age from 3 to 79 years) who underwent adrenalectomy at Hamamatsu University Hospital in Hamamatsu, Ja- pan. Forty-eight were samples of adrenal tumor tis- sues and 27 were samples of adjacent normal adrenal tissue. According to the clinical and postoperative pathologic examinations, 45 were diagnosed as benign and 3 were diagnosed as malignant. The postoperative pathologic diagnoses of all 48 patients are shown in Table 1. Pathologic examination revealed three malig- nant tumors: one functioning adrenocortical cancer, one nonfunctioning adrenocortical cancer, and one adrenal metastasis from lung carcinoma. The other 45 samples were diagnosed as benign.
Informed consent was obtained from all patients for analyzing the samples. All samples were immedi- ately frozen and stored at -80℃. Telomerase activity
was analyzed by the TRAP (telomerase repeat ampli- fication protocol) assay method.6 The samples were added to 30-50 uL of ice-cold TRAP lysis buffer (10 mM Tris HCl [pH 7.5], 1 mM MgCl2, 1 mM ethyleneg- lycoltetraacetic acid [EGTA], 0.1M phenylmethylsulfo- nyl fluoride, 5 mM -mercaptoethanol, 0.5% CHAPS, and 10% glycerol), depending on the size of the tissue samples, and incubated for 30 minutes on ice. The lysate was centrifuged at 15,000 rpm for 30 minutes at 4℃. The supernatant fluid was added to microtubes and stored at -80℃. Protein concentrations were 5-10 mg/mL. Two uL of a CHAPS cell extract was added to a 47 µL reaction solution containing 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween-20, 1 mM EGTA, 50 µM diethylnitrophenyl thiophosphate, 1 µg T4 gene 32 protein (Boehringer Mannheim, Germany), 2 units Taq DNA polymerase (Takara, Japan), bovine serum albumin (0.1 mg/mL), and 0.1 µg TS primer (5’-AATCCGTCGAGCAGAGTT- 3’) for 30 minutes at 23℃ and then heated to 95℃ for 1 minute. With 0.1 µg CX primer (5’-CCCTTACCCT- TACCCTTACCCTAA-3’), the reaction mixture was am- plified for 35 cycles at 94℃ for 30 seconds, 55℃ for 30 seconds, and 72℃ for 90 seconds, and then continu- ously incubated for 7 minutes at 72℃. The polymerase chain reaction (PCR) product was analyzed in 5% aga- rose gels (Nusieve 3:1 agarose; FMC Bio Products, Rockland, ME) in Tris-borate buffer (89 mM [pH 8.0]) containing 2 mM ethylenediamine tetraacetic acid. Ten uL of PCR product was applied to a 2-mm-thick slab gel spread onto a plate (10 × 10 cm). The current was run at 30mA for 45 minutes in the same buffer. The gel was then stained with ethidium bromide and observed under UV illumination. In all of the positive telomerase activity and/or cancer samples, the speci- ficity of the extensions was confirmed by using RNase (Boehringer Manheim, GmbH, Germany). One AL RNase was added to the 1 µg of sample and incubated 30°℃ for 10 minutes. Telomerase activity was mea- sured after the incubation.
RESULTS
Telomerase activity was detected in 7 (14.6%) of the 48 tumor tissue samples (Table 1, Fig. 1). In these seven samples, three were diagnosed as malignant after the pathologic examinations: one was nonfunctioning ad- renocortical cancer, another was functioning adreno- cortical cancer, and the third was adrenal metastasis from lung carcinoma. The other four tumors were diagnosed as benign: two were cortical adenoma, one was cortical hyperplasia, and one was pheochromocy- toma. Telomerase activity was not observed in any of the 27 samples of normal adrenal tissue.
In our results, larger tumors tended to show te-
Marker
Case 1
Case 2
P. C.
6
2
RNase
(-) (+) (-) (+) (-) (+) (-) (+)
40bp
lomerase activity. Four of 13 large tumors larger than 5 cm (30.8%) showed positive telomerase activity. However, positive telomerase activity was also de- tected in 3 of 35 tumors smaller than 5 cm (8.6%).
Telomerase positive patients are listed in Table 2. Case 1 was a male age 62 years.23 His lung carcinoma was removed 15 months previously and had been treated with radiotherapy and chemotherapy. How- ever, he was found to have a metastatic lesion in the right adrenal gland. The adrenal tissue that was re- moved contained poorly differentiated adenocarci- noma, which was consistent with the previously ex- cised lung carcinoma. The patient died 8 months later from widespread metastases.
Case 2 was a female age 43 years who visited our hospital with a report of progressive weight loss. A huge retroperitoneal mass was found above the left kidney on a computed tomogram. The tumor size was approximately 12 cm. According to an endocrinologic study, the tumor seemed to be nonfunctioning. Patho- logic examination revealed many atypical cells with
mitosis, which was consistent with adrenocortical cancer. No recurrence was found 9 months after the surgery.
Case 3 was a female infant age 3 years with ab- normal external genitalia. By endocrinologic and im- aging studies, she was diagnosed as having a testos- terone-producing adrenal tumor, and adrenalectomy was performed. According to Weiss’s criteria,24 the tumor showed nuclear atypia; clear cytoplasm; diffuse architecture; and vascular, capsular, and sinusoidal invasions. Telomerase activity was positive but weak, and no recurrence appeared after the surgery.
Case 4 was a female age 50 years.25 After an as- sessment of clinical symptoms, an endocrinologic study, and imaging examinations, she was diagnosed with Cushing’s syndrome. Nineteen months after sur- gery, multiple intraperitoneal metastases were found. At that time, a detailed pathologic reevaluation was performed. According to Weiss’s nine criteria,24 it was considered to be the functioning adrenocortical can- cer. Fourteen months after the second surgery, she died from the cancer.
Case 5 was a female age 37 years who was referred to our hospital for an investigation of the genesis of hypertension. According to an endocrinologic study and imaging studies, she was diagnosed as having pheochromocytoma and underwent surgery. How- ever, a small retroperitoneal mass appeared on a com- puted tomogram 7 months after adrenalectomy, and it was gradually growing. Serum cathecholamine levels were also increased again. We considered it a local recurrence of pheochromocytoma, and reoperation will be performed shortly.
The other two telomerase positive patients are alive and well. No recurrences have appeared.
DISCUSSION
It is thought that about 10% of pheochromocytomas, which arise from the adrenal medulla, have malignant potential.26,27 Adrenocortical tumors that are very large and/or metastasize may be easy to diagnose as malignant. However, for most adrenal tumors in adults, malignant potential is difficult to predict even with detailed endocrinologic and imaging studies. Thus, we have to depend on pathologic examination. Disappointingly, it has been difficult even for skilled pathologists to discriminate cancer from adenoma by microscopic examination. Weiss24 advocated the use of a multifactorial analysis of nine criteria to differen- tiate metastasizing tumors from nonmetastasizing tu- mors. Although his criteria is a valuable method for pathologic diagnosis, it is not easy and not generally used. The aneuploid pattern in DNA flow cytometry is also useful as a method to predict the potential bio-
| Patient no. | Age (yrs) | Gender | Tumor size (cm) | Postoperative pathologic diagnosis | Telomerase | Prognosis | |
|---|---|---|---|---|---|---|---|
| Cell type | Cancer | ||||||
| 1 | 62 | M | 5.5 | Metastatic cancer | Yes | + | Died, 8 mos. |
| 2 | 43 | F | 12.0 | Nonfunctioning cortical cancer | Yes | + | No recurrence, 17 mos. |
| 3 | 3 | F | 3.7 | Functioning cortical cancer | Yes | + | No recurrence, 72 mos. |
| 4 | 50 | F | 5.0 | Cortical adenoma | No | + | Died, 33 mos. |
| Possible recurrence, 6 | |||||||
| 5 | 37 | F | 3.5 | Pheochromocytoma | No | + | mos. |
| 6 | 40 | M | 1.9 | Cortical adenoma | No | + | No recurrence, 12 mos. |
| 7 | 63 | M | 5.5 | Cortical hyperplasia | No | + | No recurrence, 50 mos. |
logic activity of large adrenal cortical tumors.28,29 By this method, most malignant adrenal tumors were reported to show the aneuploid pattern (in contrast to benign tumors, which showed the diploid pat- tern).28,29 However, malignant adrenal tumors often had a normal diploid pattern.
In recent years, the telomerase activity of malig- nant tumors in various organs has been reported.6-16 According to the theory of cell proliferation, tumor cells with positive telomerase activity can have infinite cell division. Even cells with otherwise normal char- acteristics must be considered clinically malignant if the cells have unrestricted cell division. Thus, the de- tection of telomerase activity might be a valuable tool for predicting the malignant potential of adrenal tu- mors.
Of our seven telomerase positive patients, one had metastasis from lung carcinoma and another two had tumors clinically diagnosed as malignant before adre- nalectomy. In the other four patients, it was difficult to predict the malignant potential of the tumors before surgery, after routine pathologic examination. One of the tumors recurred, and the patient died; in another patient, a possible recurrence has appeared. Even though the tumor seems to be histologically benign, it should be considered clinically malignant when the tumor has the potential for infinite proliferation. The follow-up period has been short for two other patients with no recurrences, and we must carefully follow these telomerase positive patients. If the tumor was not completely removed, they have the risk of recur- rence.
Solid, large adrenal tumors more than 3-6 cm in size should be removed, considering the possibility of malignancy.28,29 Our results confirmed that larger tu- mors have a higher rate of telomerase activity. How- ever, telomerase activity was also detected in small tumors. We consider that patients with small but te- lomerase positive tumors are fortunate to have their
tumors removed early. For this reason, adrenalectomy should be performed even on a small tumor if the patient is willing to undergo the surgery, because we have no effective drug to treat adrenal cancer.
TRAP assay of telomerase is easy to perform, and it is valuable to know the potential for malignancy in adrenal tumors. When telomerase activity is positive and radical surgery is not performed, careful follow-up is needed.
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