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Renal cell carcinoma (RCC) and telomerase activity: relationship to stage
Urologic Oncology: Seminars and Original Investigations 21 (2003) 424 – 430
Original article
Renal cell carcinoma (RCC) and telomerase activity: relationship to stage Tarek M. Mekhail, M.D.a,*, Rika Kawanishi-Tabata, M.D.a, Raymond Tubbs, M.D.b, Andrew Novick, M.D.c, Paul Elson, DSca, Ram Ganapathi, Ph.D.a, Mahrukh Ganapathi, Ph.D.a, Ronald Bukowski, M.D.a a
Expermental Therapeutics, Taussig Cancer Center, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 b Department of Clinical Pathology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 c Urological Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 Received 31 August 2002; received in revised form 5 December 2002; accepted 3 January 2003
Abstract Limited information is available on the correlation of telomerase activity and the clinical and pathological characteristics, in patients with renal cell carcinoma (RCC). Telomerase repeat amplification protocol (TRAP) was used to measure telomerase activity in frozen RCC specimens from partial/radical nephrectomies performed between 1987 and 1991. Presence of tumor tissue was verified by a pathologist using hematoxylin and eosin stained sections. RNA was measured to ensure the presence of intact protein necessary for telomerase expression. Data on demographics, tumor type, and stage at presentation, local recurrence, distant metastasis, disease-free survival (DFS), and overall survival (OS) was collected, and telomerase activity was correlated with each of these variables. Forty-nine of 67 patients (73%) were telomerase positive (⫹ve). Gender and stage were the only variables that appeared to be associated with telomerase positivity. Tumors were telomerase ⫹ve in 12/21 females (57 %) vs. 37/46 males (80%) (P ⫽ 0.07). Tumors were telomerase ⫹ve in 85% of Stage IV, 76% of Stage III, and 70% of Stage I/II patients (P ⫽ 0.12). Five-year survival was 0% for Stage IV, 57% for Stage III, and 77% for Stage I/II patients (P ⬍ 0.001), DFS 54% for stage III and 84% for Stage I/II patients (P ⫽ 0.05). Telomerase activity, however, was not related to survival in either univariate or multivariate analysis. In patients with telomerase ⫹ve tumors 5-year survival was 55%, and with telomerase ⫺ve tumors 58% (P ⫽ 0.56). Stage was the only variable associated with OS or DFS in clear cell RCC patients. In patients with advanced disease, there is a high incidence of telomerase positivity was found, within this limited sample, however, no correlation with survival was found. © 2003 Elsevier Inc. All rights reserved. Keywords: Telomerase; Renal cell carcinoma
1. Introduction Telomerase is an important enzyme whose activity has been convincingly demonstrated in humans recently. It is required for maintenance of ends of chromosomes (telomeres) during cell division. During each cycle of cell division telomeres are progressively eroded until they reach a length that coincides with the activation of an antiproliferative mechanism termed mortality stage 1 (M1). Thus, telomere shortening functions as a ‘mitotic clock’ and limits division in somatic cells, thereby contributing to cellular * Corresponding author Tel: ⫹1-216-445-1785; fax: ⫹1-216-4449464. E-mail address: [email protected] (T.M. Mekhail). 1078-1439/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1078-1439(02)00003-6
aging. Telomerase can compensate for the loss of telomeric repeats during cell division, thus contributing to immortalization of cells [1]. Development of the TRAP (telomerase repeat amplification protocol) assay, by Kim et al. [2] has opened the door to large scale testing for telomerse activity in various types of tumors. Using this method, a variety of cell lines and malignant tumors have been found to express telomerase activity, in contrast to most human somatic cells that express low or undetectable levels of telomerase activity. These findings suggest that telomerase activation may be a critical step in cell immortalization and oncogenesis. Telomerase activity has been previously demonstrated in renal cell carcinoma [3,4]; however, limited information is available on its correlation with the clinical and pathological characteristics in this patient group. We conducted this
T.M. Mekhail et al. / Urologic Oncology: Seminars and Original Investigations 21 (2003) 424 – 430
study to further investigate the expression of telomerase activity in renal cell cancer, as well as its association with a variety of clinical variables.
2. Methods We identified patients who underwent radical or partial nephrectomy for a diagnosis of clear cell renal cell carcinoma between the years of 1987 and 1991, for whom frozen tumor tissue collected at the time of surgery was stored. This time period was selected to ensure at least 5-year follow up period at the time this study was undertaken. 2.1. Tissue samples Tissue biopsies obtained at the time of surgery, were snap frozen in liquid nitrogen and stored at ⫺80°C. For determination of telomerase activity and for rRNA analysis 20 –30 ribbons of five micron cryosections were homogenized by vortexing in 0.3 mL of CHAPS lysis buffer (10 mM Tris-HCL, ph 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM Benzamidine, 5 mM -mercaptoethanol, 0.5% CHAPS and 10% glycerol). The extracts were incubated at 4°C for 30 min and centrifuged at 12,000 ⫻ g, 4°C for 20 min. The supernatant was stored at 80°C. 2.2. Telomerase assay Telomerase activity was determined with the TRAP-eze telomerase detection kit (TRAPeze, Intergen). The procedure was carried out as described in the manufacturer’s protocol [5], with minor modifications, as described by Kawanishi-Tabata et al. [6] Each sample was assayed in duplicate. In addition, a heat inactivated negative control assay was performed following incubation of the tissue extract at 85°C for 10 min. Telomere extension was first carried out at 30°C for 1 h in a separate reaction (total volume of 20 L) containing 4 L of tissue extract. Following dilution of the reaction mixture to 40 mL with Tris-EDTA (TE) buffer, the telomeric DNA was extracted with phenol:chloroform. An aliquot (2 mL) of the aqueous phase containing the extended telomeric DNA was amplified by PCR (1 cycle of 30°C for 30 min, 94°C for 5 min, 35 cycles of 94°C for 30 sec, 52°C for 30 sec and 72°C for 45 sec, followed by a final cycle of 72°C for 5 min). This aliquot corresponds to 0.2 mL of the original tissue extract. A positive quantitation control (0.1 amoles of TSR8 DNA) and a negative buffer control were included with each PCR reaction. The PCR products (15 L) were analyzed by gel electrophoresis on 12% polyarcylamide nondenutring gels in TBE buffer. The gels were dried and the radioactivity present in the samples was determined by Phosphoimage analysis of the telomeric ladder. Telomerase activity was scored positive if the intensity of the sample lane was greater than that of the negative control lane. Tissue sam-
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ples, in which telomerase activity was not detected, were re-analyzed using ten times more sample. For this purpose the reaction mixture containing the extended telomeric DNA was diluted to 100 mL with TE buffer and extracted with phenol:chloroform. An aliquot (50 mL) of the aqueous phase was precipitated with ethanol and used for the PCR reaction. This aliquot corresponds to 2 mL of the original tissue extract. 2.3. Determination of rRNA content in tissue samples The levels of 28S rRNA were measured using the EZ RNA PCR kit essentially as described by Clark et al. [7] This assay was used to ascertain the integrity of tissue samples. Absence of telomerase mRNA and activity and rRNA in frozen samples would suggest degradation of the tissue, and these samples were excluded from the analysis. All 67 samples included in the analysis contained intact rRNA. 2.4. Clinical and pathological data Patients’ records were reviewed and demographics, clinical data, TNM stage [8] and histologic type and grade, pattern of tumor recurrence (local versus systemic), disease free survival and overall survival were recorded. 2.5. Statistical analysis Telomerase activity determined on tumor tissue samples determined using the TRAP assay was dichotomized as Table 1 Patients with renal cell carcinoma*: distribution of telomerase activity in various clinical groups
Sex Female Male Age ⱕ55 ⬎55 Median (range) Stage I/II III IV Bilateral Disease No Yes
Telomerase (⫺)
Telomerase (⫹ ve)
Overall
N ⫽ 18
N ⫽ 49
N ⫽ 67
12 (57%) 37 (80%)
21 (32%) 46 (68%)
22 (80%) 27 (67%) 60 (32–87)
27 (40%) 40 (60%) 61 (32–87)
11 (30%) 4 (24%) 2 (15%)
26 (70%) 13 (76%) 11 (85%)
37 (55%) 17 (25%) 13 (20%)
16 (29%) 4 (33%)
39 (71%) 8 (67%)
55 (82%) 12 (18%)
P-value
0.07 9 (43%) 9 (20%)
0.27 5 (20%) 13 (33%) 63.5 (48–80)
0.38
0.74
* Includes 62 patients with clear cell carcinoma, 2 patients with mixed granular cell and clear cell histology, 1 papillary and 1 granular cell histology.
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T.M. Mekhail et al. / Urologic Oncology: Seminars and Original Investigations 21 (2003) 424 – 430
Fig. 1. Telomerase activity in tissue extracts from 8 patients with Renal Cell Cancer: In this representative experiment cell extracts from tumor specimens were assayed for telomerase activity using the telomerase repeat amplification protocol. Each sample was assayed in duplicate and an additional assay of the sample was performed after heat inactivation (H). A negative CHAPS buffer control and telomerase quantitation control template (TSR8) also were included in each experiment. Bp: base pairs.
positive or negative as described above. The association between telomerase activity and clinico-pathological variables such as gender, age and stage was evaluated using Fisher’s exact test and the Cochran-Armitage test [9]. The method of Kaplan and Meier was used summarize the distributions of overall survival and disease free survival, and the log-rank test was used to evaluate individual factors with respect to these outcomes. The Cox proportional hazards model was used to simultaneously assess multiple factors. Overall survival was measured from initial diagnosis to death, or date last known to be alive. Disease free survival was measured from initial diagnosis to recurrence. Patients who died without evidence of recurrence were censored as of the date of death.
3. Results Sixty-seven patients with renal cell carcinoma were identified. Sixty-two patients had clear cell carcinoma, and two mixed granular cell and clear cell histology. Three of 67 had granular (two) and papillary (one) carcinoma, and were excluded from analysis. Forty-nine of 67 specimens (73%) were telomerase positive. Three of the 4 samples with gran-
ular cell component, and the one papillary carcinoma sample were telomerase positive. Table 1 summarizes patient characteristics. Gender and stage were the only two variables that appeared to be related to telomerase positivity. Tumors were telomerase positive in 12 of 21 females (57%) vs. 37 of 46 males (80%), P ⫽ .07. Eighty-five percent of Table 2 Clear cell renal cell carcinoma: correlation of overall survival with telomerase expression and clinical variables (n ⫽ 58)
Telomerase Negative Positive Sex Female Male Age ⱕ55 ⬎55 Stage I/II III IV
Number (deaths)
2-year survival
5-year survival
15 (8) 43 (26)
79% 67%
57% 55%
18(8) 40 (26)
83% 64%
72% 48%
25 (16) 33 (18)
54% 82%
42% 66%
32 (3) 14 (9) 12 (12)
90% 71% 17%
77% 57% 0%
P-value
0.56 0.07
0.18
⬍0.001
T.M. Mekhail et al. / Urologic Oncology: Seminars and Original Investigations 21 (2003) 424 – 430 Table 3 Clear cell renal cell carcinoma: correlation of disease free survival (DFS) with telomerase expression and clinical variables (Stage I–III, N ⫽ 45)
Telomerase Negative Positive Sex Female Male Age ⱕ55 ⬎55 Stage I/II III
Number (number of failures)
2-year DFS
5-year DFS
14 (5) 31 (7)
78% 81%
69% 79%
17 (4) 28 (8)
82% 78%
77% 74%
17 (7) 28 (5)
65% 89%
58% 85%
32 (6) 13 (6)
87% 62%
84% 54%
P-value 0.39
0.60
0.06
0.05
Stage IV patients and 76% of Stage III patients were telomerase positive, compared to 70% of Stage I/II patients, P ⫽ .38. It must be noted, however, that gender and stage were also related. Seventy-one percent (15/21) of females were Stage I or II and 5% (1/21) were Stage IV, compared to 48% (22/46) and 26% (12/46), respectively, for males, P ⫽ 0.04.
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Twelve patients had either synchronous or metachronous bilateral cell carcinoma. Eight of 12 (67%) were telomerase positive, P ⫽ .74. A representative experiment is shown in Fig. 1, in which tissue extract from 8 renal cell cancer patients were analyzed for telomerase activity. Table 2 summarizes overall survival data and Table 3 disease free survival data. Overall 34 of 58 patients in whom survival data were available have expired. The estimated 2 and 5-year survival rates are 74% and 56%, respectively. Not surprisingly, both univariate and multivariate analysis identified stage as a predictor of survival. Patients with Stage I or II disease have estimated 2-year survival of 90% compared to 71% for Stage III and 17% for Stage IV disease, P ⬍ .001. Overall survival did not correlate with telomerase activity (Fig. 2). Considering that most patients (11 of 13) with Stage IV disease were telomerase positive, and have poor prognosis, the possibility this would mask differences was considered. Therefore, only Stages I, II and III were analyzed, however, telomerase activity still failed to correlate with survival (P ⫽ .65). Forty-five patients with Stages I to III had data regarding time to disease recurrence available. Twelve of 45 patients had recurred with estimated two and 5-year disease free survival rates of 80% and 75%, respectively. Ten of 12
Fig. 2. Overall survival by telomerase expression.
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Fig. 3. Disease free survival by telomerase expression: Stages I–III.
individuals developed distant metastatic sites, 1 a local recurrence and 1 patient expired with evidence of disease, but the site was not specified. As with overall survival, stage was associated with disease free survival in both univariate (P ⫽ .05) and multivariate (P ⫽ .05) analysis. No association between telomerase activity and disease free survival was found in this group (Fig. 3).
4. Discussion Over the last 7 years, screening of most types of human cancer has established a very strong association between telomerase activity and malignancy, making the enzyme one of the most common tumor markers [2,10,11]. Shay et al. reported that 758 of 895 (85%) tissue samples from different malignant tumors, and none of 70 normal somatic tissues, expressed telomerase activity [11]. Telomerase activity has previously been reported in renal cell carcinoma, with an incidence of 60% to 92% reported in various studies [3,12–14]. In the current series 49 of 67 tumor specimens were telomerase positive, an incidence of 73%, and is consistent with previous reports. A possible explanation for the presence of telomerase negative tumors includes alternate mechanisms in these tumors for maintaining telomere length, and preventing telomeres from reaching the critical length and the subsequent timed cell death. Recent reports by Murnane et al. [15] and Bryan et al. [16] demonstrated a comparable telomerase-independent mechanism in an immortalized human cell that does not contain detectable te-
lomerase activity. Mechanisms of cellular immortality, therefore, may not be limited to telomerase activation, and alternative pathways to cellular immortality may exist. Telomere elongation by telomere recombination [17,18] is one example of such alternate mechanisms. Other explanations for a telomerase negativity include, sample integrity and the absence of tumor tissue in the sample tested (sample error). In the current study 28S rRNA levels were measured in all samples, and those in which it was undetectable and were telomerase negative, were considered degenerated and excluded from the analysis (number 20). Hematoxylin and eosin stained sections of all samples were reviewed by a pathologist to ensure the presence of tumor tissue. One final explanation for false telomerase negativity, is the presence of Taq polymerase inhibitors that might interfere with the polymerase chain reaction (PCR) [19,20]. Recent studies have reported a positive correlation between telomerase activity and clinical stage or pathological grade in some tumor types [21–23]. The current study demonstrated a trend towards the association of telomerase activity and stage. Eleven of 13 (85%) Stage IV tumors were telomerase positive compared to 70% of Stage I/II tumors. A larger sample size may be required to clearly demonstrate this correlation. Additionally, no correlation between survival and telomerase activity, using either univariate or multivariate analyses, was found. This is consistent with previously reported studies. In the report by Sugimura et al. [12], 53 tumors from 52 patients were studied. Twenty-five of 38 clear cell carcinomas were telomerase positive (66%), and 5 of 9 granular cell carcinomas were
T.M. Mekhail et al. / Urologic Oncology: Seminars and Original Investigations 21 (2003) 424 – 430
positive. In this report no association, with clinical stage, tumor size, grade or pathological type was demonstrated. A similar finding was also reported by Mehle et al. [3], who studied forty RCC (histological type not specified) tumors. Seventy-one percent of which were telomerase positive. Fujioka et al. [13], examined 30 RCC neoplasms and reported 12 of 21 (57%) clear cell carcinomas were telomerase positive. As in other reports, no correlation with tumor size, stage, histological subtype or DNA ploidy were noted, however, they noted a statistically significant correlation between tumor grade and telomerase activity [13]. Thirtyeight percent of Grade 1 tumors were telomerase positive compared to 76.5% of Grade 2–3 tumors, P ⫽ .05 [13]. Kinoshita et al. [14] in a study of 47 RCC samples, reported telomerase activity in 36 (77%), however only one of six chromphobe tumor was telomerase positive (P ⬍ .001). In contrast 26 of 28 (93%) clear cell carcinoma tumors, and three of four granular cell subtype were telomerase positive [14]. The correlation of telomerase activity and patient outcome was not reported in this study [14]. In the current series three of four granular cell tumors were telomerase positive, and no chromphobe tumors were studied. Conversely, studies of telomerase activity in other kinds of malignancies including, neuroblastoma [24], acute myeloid leukemia [10,25], breast [26,27] and gastrointestinal cancers prognostic [21,28] suggests a possible correlation of telomerse activity and poor prognosis. In a report by Hiyama et al. [24] the correlation of telomerase activity with outcome for neuroblastoma patients was examined. Ninetyfive of 100 neuroblastoma cases were telomerase positive. Tumors with high telomerase activity had genetic alterations (e.g., N-myc amplification) and an unfavorable prognosis, whereas tumors with lower telomerase activity were devoid of these genetic changes and were associated with a more favorable prognosis. Of most interest is the finding that the three neuroblastomas that lacked telomerase activity, underwent spontaneous regressions. These findings are intriguing, and although similar observations have not been made in renal cell cancer, the finding by Kinoshita et al. [14] that telomerase activity in the chromphobe subtype of renal cancer is absent or low is of interest in view of its known less aggressive behavior [29,30]. Further investigation of this may be warranted. Also, renal cell cancer is a malignancy where spontaneous regressions have been reported [31,33]. Investigation of telomerase activity in these patients as well as those who respond to therapy would be of interest. In conclusion, it is possible that telomerase activity may have varying roles in different malignancies. Its use as a prognostic factor, as a predictor of response to therapy, and as a target of therapy require further study.
Acknowledgments Supported by grant from GERON Corporation.
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Original article
Renal cell carcinoma (RCC) and telomerase activity: relationship to stage Tarek M. Mekhail, M.D.a,*, Rika Kawanishi-Tabata, M.D.a, Raymond Tubbs, M.D.b, Andrew Novick, M.D.c, Paul Elson, DSca, Ram Ganapathi, Ph.D.a, Mahrukh Ganapathi, Ph.D.a, Ronald Bukowski, M.D.a a
Expermental Therapeutics, Taussig Cancer Center, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 b Department of Clinical Pathology, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 c Urological Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 Received 31 August 2002; received in revised form 5 December 2002; accepted 3 January 2003
Abstract Limited information is available on the correlation of telomerase activity and the clinical and pathological characteristics, in patients with renal cell carcinoma (RCC). Telomerase repeat amplification protocol (TRAP) was used to measure telomerase activity in frozen RCC specimens from partial/radical nephrectomies performed between 1987 and 1991. Presence of tumor tissue was verified by a pathologist using hematoxylin and eosin stained sections. RNA was measured to ensure the presence of intact protein necessary for telomerase expression. Data on demographics, tumor type, and stage at presentation, local recurrence, distant metastasis, disease-free survival (DFS), and overall survival (OS) was collected, and telomerase activity was correlated with each of these variables. Forty-nine of 67 patients (73%) were telomerase positive (⫹ve). Gender and stage were the only variables that appeared to be associated with telomerase positivity. Tumors were telomerase ⫹ve in 12/21 females (57 %) vs. 37/46 males (80%) (P ⫽ 0.07). Tumors were telomerase ⫹ve in 85% of Stage IV, 76% of Stage III, and 70% of Stage I/II patients (P ⫽ 0.12). Five-year survival was 0% for Stage IV, 57% for Stage III, and 77% for Stage I/II patients (P ⬍ 0.001), DFS 54% for stage III and 84% for Stage I/II patients (P ⫽ 0.05). Telomerase activity, however, was not related to survival in either univariate or multivariate analysis. In patients with telomerase ⫹ve tumors 5-year survival was 55%, and with telomerase ⫺ve tumors 58% (P ⫽ 0.56). Stage was the only variable associated with OS or DFS in clear cell RCC patients. In patients with advanced disease, there is a high incidence of telomerase positivity was found, within this limited sample, however, no correlation with survival was found. © 2003 Elsevier Inc. All rights reserved. Keywords: Telomerase; Renal cell carcinoma
1. Introduction Telomerase is an important enzyme whose activity has been convincingly demonstrated in humans recently. It is required for maintenance of ends of chromosomes (telomeres) during cell division. During each cycle of cell division telomeres are progressively eroded until they reach a length that coincides with the activation of an antiproliferative mechanism termed mortality stage 1 (M1). Thus, telomere shortening functions as a ‘mitotic clock’ and limits division in somatic cells, thereby contributing to cellular * Corresponding author Tel: ⫹1-216-445-1785; fax: ⫹1-216-4449464. E-mail address: [email protected] (T.M. Mekhail). 1078-1439/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1078-1439(02)00003-6
aging. Telomerase can compensate for the loss of telomeric repeats during cell division, thus contributing to immortalization of cells [1]. Development of the TRAP (telomerase repeat amplification protocol) assay, by Kim et al. [2] has opened the door to large scale testing for telomerse activity in various types of tumors. Using this method, a variety of cell lines and malignant tumors have been found to express telomerase activity, in contrast to most human somatic cells that express low or undetectable levels of telomerase activity. These findings suggest that telomerase activation may be a critical step in cell immortalization and oncogenesis. Telomerase activity has been previously demonstrated in renal cell carcinoma [3,4]; however, limited information is available on its correlation with the clinical and pathological characteristics in this patient group. We conducted this
T.M. Mekhail et al. / Urologic Oncology: Seminars and Original Investigations 21 (2003) 424 – 430
study to further investigate the expression of telomerase activity in renal cell cancer, as well as its association with a variety of clinical variables.
2. Methods We identified patients who underwent radical or partial nephrectomy for a diagnosis of clear cell renal cell carcinoma between the years of 1987 and 1991, for whom frozen tumor tissue collected at the time of surgery was stored. This time period was selected to ensure at least 5-year follow up period at the time this study was undertaken. 2.1. Tissue samples Tissue biopsies obtained at the time of surgery, were snap frozen in liquid nitrogen and stored at ⫺80°C. For determination of telomerase activity and for rRNA analysis 20 –30 ribbons of five micron cryosections were homogenized by vortexing in 0.3 mL of CHAPS lysis buffer (10 mM Tris-HCL, ph 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM Benzamidine, 5 mM -mercaptoethanol, 0.5% CHAPS and 10% glycerol). The extracts were incubated at 4°C for 30 min and centrifuged at 12,000 ⫻ g, 4°C for 20 min. The supernatant was stored at 80°C. 2.2. Telomerase assay Telomerase activity was determined with the TRAP-eze telomerase detection kit (TRAPeze, Intergen). The procedure was carried out as described in the manufacturer’s protocol [5], with minor modifications, as described by Kawanishi-Tabata et al. [6] Each sample was assayed in duplicate. In addition, a heat inactivated negative control assay was performed following incubation of the tissue extract at 85°C for 10 min. Telomere extension was first carried out at 30°C for 1 h in a separate reaction (total volume of 20 L) containing 4 L of tissue extract. Following dilution of the reaction mixture to 40 mL with Tris-EDTA (TE) buffer, the telomeric DNA was extracted with phenol:chloroform. An aliquot (2 mL) of the aqueous phase containing the extended telomeric DNA was amplified by PCR (1 cycle of 30°C for 30 min, 94°C for 5 min, 35 cycles of 94°C for 30 sec, 52°C for 30 sec and 72°C for 45 sec, followed by a final cycle of 72°C for 5 min). This aliquot corresponds to 0.2 mL of the original tissue extract. A positive quantitation control (0.1 amoles of TSR8 DNA) and a negative buffer control were included with each PCR reaction. The PCR products (15 L) were analyzed by gel electrophoresis on 12% polyarcylamide nondenutring gels in TBE buffer. The gels were dried and the radioactivity present in the samples was determined by Phosphoimage analysis of the telomeric ladder. Telomerase activity was scored positive if the intensity of the sample lane was greater than that of the negative control lane. Tissue sam-
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ples, in which telomerase activity was not detected, were re-analyzed using ten times more sample. For this purpose the reaction mixture containing the extended telomeric DNA was diluted to 100 mL with TE buffer and extracted with phenol:chloroform. An aliquot (50 mL) of the aqueous phase was precipitated with ethanol and used for the PCR reaction. This aliquot corresponds to 2 mL of the original tissue extract. 2.3. Determination of rRNA content in tissue samples The levels of 28S rRNA were measured using the EZ RNA PCR kit essentially as described by Clark et al. [7] This assay was used to ascertain the integrity of tissue samples. Absence of telomerase mRNA and activity and rRNA in frozen samples would suggest degradation of the tissue, and these samples were excluded from the analysis. All 67 samples included in the analysis contained intact rRNA. 2.4. Clinical and pathological data Patients’ records were reviewed and demographics, clinical data, TNM stage [8] and histologic type and grade, pattern of tumor recurrence (local versus systemic), disease free survival and overall survival were recorded. 2.5. Statistical analysis Telomerase activity determined on tumor tissue samples determined using the TRAP assay was dichotomized as Table 1 Patients with renal cell carcinoma*: distribution of telomerase activity in various clinical groups
Sex Female Male Age ⱕ55 ⬎55 Median (range) Stage I/II III IV Bilateral Disease No Yes
Telomerase (⫺)
Telomerase (⫹ ve)
Overall
N ⫽ 18
N ⫽ 49
N ⫽ 67
12 (57%) 37 (80%)
21 (32%) 46 (68%)
22 (80%) 27 (67%) 60 (32–87)
27 (40%) 40 (60%) 61 (32–87)
11 (30%) 4 (24%) 2 (15%)
26 (70%) 13 (76%) 11 (85%)
37 (55%) 17 (25%) 13 (20%)
16 (29%) 4 (33%)
39 (71%) 8 (67%)
55 (82%) 12 (18%)
P-value
0.07 9 (43%) 9 (20%)
0.27 5 (20%) 13 (33%) 63.5 (48–80)
0.38
0.74
* Includes 62 patients with clear cell carcinoma, 2 patients with mixed granular cell and clear cell histology, 1 papillary and 1 granular cell histology.
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Fig. 1. Telomerase activity in tissue extracts from 8 patients with Renal Cell Cancer: In this representative experiment cell extracts from tumor specimens were assayed for telomerase activity using the telomerase repeat amplification protocol. Each sample was assayed in duplicate and an additional assay of the sample was performed after heat inactivation (H). A negative CHAPS buffer control and telomerase quantitation control template (TSR8) also were included in each experiment. Bp: base pairs.
positive or negative as described above. The association between telomerase activity and clinico-pathological variables such as gender, age and stage was evaluated using Fisher’s exact test and the Cochran-Armitage test [9]. The method of Kaplan and Meier was used summarize the distributions of overall survival and disease free survival, and the log-rank test was used to evaluate individual factors with respect to these outcomes. The Cox proportional hazards model was used to simultaneously assess multiple factors. Overall survival was measured from initial diagnosis to death, or date last known to be alive. Disease free survival was measured from initial diagnosis to recurrence. Patients who died without evidence of recurrence were censored as of the date of death.
3. Results Sixty-seven patients with renal cell carcinoma were identified. Sixty-two patients had clear cell carcinoma, and two mixed granular cell and clear cell histology. Three of 67 had granular (two) and papillary (one) carcinoma, and were excluded from analysis. Forty-nine of 67 specimens (73%) were telomerase positive. Three of the 4 samples with gran-
ular cell component, and the one papillary carcinoma sample were telomerase positive. Table 1 summarizes patient characteristics. Gender and stage were the only two variables that appeared to be related to telomerase positivity. Tumors were telomerase positive in 12 of 21 females (57%) vs. 37 of 46 males (80%), P ⫽ .07. Eighty-five percent of Table 2 Clear cell renal cell carcinoma: correlation of overall survival with telomerase expression and clinical variables (n ⫽ 58)
Telomerase Negative Positive Sex Female Male Age ⱕ55 ⬎55 Stage I/II III IV
Number (deaths)
2-year survival
5-year survival
15 (8) 43 (26)
79% 67%
57% 55%
18(8) 40 (26)
83% 64%
72% 48%
25 (16) 33 (18)
54% 82%
42% 66%
32 (3) 14 (9) 12 (12)
90% 71% 17%
77% 57% 0%
P-value
0.56 0.07
0.18
⬍0.001
T.M. Mekhail et al. / Urologic Oncology: Seminars and Original Investigations 21 (2003) 424 – 430 Table 3 Clear cell renal cell carcinoma: correlation of disease free survival (DFS) with telomerase expression and clinical variables (Stage I–III, N ⫽ 45)
Telomerase Negative Positive Sex Female Male Age ⱕ55 ⬎55 Stage I/II III
Number (number of failures)
2-year DFS
5-year DFS
14 (5) 31 (7)
78% 81%
69% 79%
17 (4) 28 (8)
82% 78%
77% 74%
17 (7) 28 (5)
65% 89%
58% 85%
32 (6) 13 (6)
87% 62%
84% 54%
P-value 0.39
0.60
0.06
0.05
Stage IV patients and 76% of Stage III patients were telomerase positive, compared to 70% of Stage I/II patients, P ⫽ .38. It must be noted, however, that gender and stage were also related. Seventy-one percent (15/21) of females were Stage I or II and 5% (1/21) were Stage IV, compared to 48% (22/46) and 26% (12/46), respectively, for males, P ⫽ 0.04.
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Twelve patients had either synchronous or metachronous bilateral cell carcinoma. Eight of 12 (67%) were telomerase positive, P ⫽ .74. A representative experiment is shown in Fig. 1, in which tissue extract from 8 renal cell cancer patients were analyzed for telomerase activity. Table 2 summarizes overall survival data and Table 3 disease free survival data. Overall 34 of 58 patients in whom survival data were available have expired. The estimated 2 and 5-year survival rates are 74% and 56%, respectively. Not surprisingly, both univariate and multivariate analysis identified stage as a predictor of survival. Patients with Stage I or II disease have estimated 2-year survival of 90% compared to 71% for Stage III and 17% for Stage IV disease, P ⬍ .001. Overall survival did not correlate with telomerase activity (Fig. 2). Considering that most patients (11 of 13) with Stage IV disease were telomerase positive, and have poor prognosis, the possibility this would mask differences was considered. Therefore, only Stages I, II and III were analyzed, however, telomerase activity still failed to correlate with survival (P ⫽ .65). Forty-five patients with Stages I to III had data regarding time to disease recurrence available. Twelve of 45 patients had recurred with estimated two and 5-year disease free survival rates of 80% and 75%, respectively. Ten of 12
Fig. 2. Overall survival by telomerase expression.
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Fig. 3. Disease free survival by telomerase expression: Stages I–III.
individuals developed distant metastatic sites, 1 a local recurrence and 1 patient expired with evidence of disease, but the site was not specified. As with overall survival, stage was associated with disease free survival in both univariate (P ⫽ .05) and multivariate (P ⫽ .05) analysis. No association between telomerase activity and disease free survival was found in this group (Fig. 3).
4. Discussion Over the last 7 years, screening of most types of human cancer has established a very strong association between telomerase activity and malignancy, making the enzyme one of the most common tumor markers [2,10,11]. Shay et al. reported that 758 of 895 (85%) tissue samples from different malignant tumors, and none of 70 normal somatic tissues, expressed telomerase activity [11]. Telomerase activity has previously been reported in renal cell carcinoma, with an incidence of 60% to 92% reported in various studies [3,12–14]. In the current series 49 of 67 tumor specimens were telomerase positive, an incidence of 73%, and is consistent with previous reports. A possible explanation for the presence of telomerase negative tumors includes alternate mechanisms in these tumors for maintaining telomere length, and preventing telomeres from reaching the critical length and the subsequent timed cell death. Recent reports by Murnane et al. [15] and Bryan et al. [16] demonstrated a comparable telomerase-independent mechanism in an immortalized human cell that does not contain detectable te-
lomerase activity. Mechanisms of cellular immortality, therefore, may not be limited to telomerase activation, and alternative pathways to cellular immortality may exist. Telomere elongation by telomere recombination [17,18] is one example of such alternate mechanisms. Other explanations for a telomerase negativity include, sample integrity and the absence of tumor tissue in the sample tested (sample error). In the current study 28S rRNA levels were measured in all samples, and those in which it was undetectable and were telomerase negative, were considered degenerated and excluded from the analysis (number 20). Hematoxylin and eosin stained sections of all samples were reviewed by a pathologist to ensure the presence of tumor tissue. One final explanation for false telomerase negativity, is the presence of Taq polymerase inhibitors that might interfere with the polymerase chain reaction (PCR) [19,20]. Recent studies have reported a positive correlation between telomerase activity and clinical stage or pathological grade in some tumor types [21–23]. The current study demonstrated a trend towards the association of telomerase activity and stage. Eleven of 13 (85%) Stage IV tumors were telomerase positive compared to 70% of Stage I/II tumors. A larger sample size may be required to clearly demonstrate this correlation. Additionally, no correlation between survival and telomerase activity, using either univariate or multivariate analyses, was found. This is consistent with previously reported studies. In the report by Sugimura et al. [12], 53 tumors from 52 patients were studied. Twenty-five of 38 clear cell carcinomas were telomerase positive (66%), and 5 of 9 granular cell carcinomas were
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positive. In this report no association, with clinical stage, tumor size, grade or pathological type was demonstrated. A similar finding was also reported by Mehle et al. [3], who studied forty RCC (histological type not specified) tumors. Seventy-one percent of which were telomerase positive. Fujioka et al. [13], examined 30 RCC neoplasms and reported 12 of 21 (57%) clear cell carcinomas were telomerase positive. As in other reports, no correlation with tumor size, stage, histological subtype or DNA ploidy were noted, however, they noted a statistically significant correlation between tumor grade and telomerase activity [13]. Thirtyeight percent of Grade 1 tumors were telomerase positive compared to 76.5% of Grade 2–3 tumors, P ⫽ .05 [13]. Kinoshita et al. [14] in a study of 47 RCC samples, reported telomerase activity in 36 (77%), however only one of six chromphobe tumor was telomerase positive (P ⬍ .001). In contrast 26 of 28 (93%) clear cell carcinoma tumors, and three of four granular cell subtype were telomerase positive [14]. The correlation of telomerase activity and patient outcome was not reported in this study [14]. In the current series three of four granular cell tumors were telomerase positive, and no chromphobe tumors were studied. Conversely, studies of telomerase activity in other kinds of malignancies including, neuroblastoma [24], acute myeloid leukemia [10,25], breast [26,27] and gastrointestinal cancers prognostic [21,28] suggests a possible correlation of telomerse activity and poor prognosis. In a report by Hiyama et al. [24] the correlation of telomerase activity with outcome for neuroblastoma patients was examined. Ninetyfive of 100 neuroblastoma cases were telomerase positive. Tumors with high telomerase activity had genetic alterations (e.g., N-myc amplification) and an unfavorable prognosis, whereas tumors with lower telomerase activity were devoid of these genetic changes and were associated with a more favorable prognosis. Of most interest is the finding that the three neuroblastomas that lacked telomerase activity, underwent spontaneous regressions. These findings are intriguing, and although similar observations have not been made in renal cell cancer, the finding by Kinoshita et al. [14] that telomerase activity in the chromphobe subtype of renal cancer is absent or low is of interest in view of its known less aggressive behavior [29,30]. Further investigation of this may be warranted. Also, renal cell cancer is a malignancy where spontaneous regressions have been reported [31,33]. Investigation of telomerase activity in these patients as well as those who respond to therapy would be of interest. In conclusion, it is possible that telomerase activity may have varying roles in different malignancies. Its use as a prognostic factor, as a predictor of response to therapy, and as a target of therapy require further study.
Acknowledgments Supported by grant from GERON Corporation.
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