High telomerase activity is associated with cell cycle deregulation and rapid progression in endometrioid adenocarcinoma of the uterus.

Telomerase activity, a mechanism granting cellular immortality, has been detected in most cancer entities, but its association with clinical, histopathologic, and prognostic parameters is not fully understood. We investigated whether quantitative telomerase levels are correlated to established prognostic factors, telomere lengths, cell cycle kinetics, and the clinical course in endometrioid adenocarcinoma of the uterus (EC). A modified telomeric repeat amplification protocol (TRAP) was used to quantify the relative telomerase activity in a series of 53 primary tumors. Mean telomere length was determined by Southern blot analysis. Cell cycle kinetics were studied immunohistochemically on paraffin sections using monoclonal antibodies to 2 distinct proliferation-specific proteins: Ki-67, which is expressed throughout the cell cycle, and a novel cell cycle-associated protein, repp86, the expression of which is restricted to the cell cycle phases S, G2, and M. The ratio of the 2 immunolabeling indices defines the rate of transition through the restriction point. Telomerase activity was detected in 50 of 53 ECs (94%). Its levels correlated significantly with FIGO stage (P =.01) and FIGO grade (P =.003) but not with myometrial invasion. They were weakly associated with the overall proliferative activity (Ki-67, r =.48) but significantly with the repp86 index (r =.64) and even more strongly with the repp86:Ki-67 ratio (r =.77). There was no correlation with mean telomere length. In the group of tumors with high telomerase activity, 5 patients had relapses and 2 died of the disease within a median follow-up period of 29 months. Recurrence showed no relation to FIGO grade and stage. No events were observed in the group with low telomerase activity. In a multivariate model including tumor stage, histopathologic grade, depth of myometrial invasion, and Ki-67 indices, telomerase activity emerged as the only independent predictor of disease progression (P =.0002). It is concluded that beyond a link to proliferation, high telomerase activity reflects a deregulation of the cell cycle associated with an increased rate of cells entering S phase and a higher degree of malignancy. Therefore, quantitative analysis of telomerase activity may be useful for identifying EC patients at high risk for recurrence.     

Telomerase activity and the risk of lung cancer

Telomerase play a key role in the maintenance of telomere length and chromosome integrity. We have evaluated the association between telomerase activity and the risk of lung cancer in peripheral blood. Telomerase activity in peripheral blood mononuclear cells was measured by a PCR-designed telomeric repeat amplification protocol in 63 lung cancer patients and 190 healthy controls that were matched for age, gender, and smoking status. Telomerase activity was significantly lower in the lung cancer patients than in controls (mean ± standard deviation; 1.32 ± 1.65 vs 2.60 ± 3.09, P < 1 × 10(-4)). When telomerase activity was categorized into quartiles based on telomerase activity in the controls, the risk of lung cancer increased as telomerase activity reduced (P(trend) = 1 × 10(-4)). Moreover, when the subjects were categorized based on the median value of telomerase activity, subjects with low telomerase activity were at a significantly increased risk of lung cancer compared to subjects with high telomerase activity (adjusted odds ratio = 3.05, 95% confidence interval = 1.60-5.82, P = 7 × 10(-4)). These findings suggest that telomerase activity may affect telomere maintenance, thereby contributing to susceptibility to lung cancer.     

Telomerase expression in intracranial tumours: prognostic potential for malignant gliomas and meningiomas.

AIM: To evaluate the diagnostic value of telomerase expression in intracranial tumours. METHODS: 98 surgical specimens from different neoplasms were analysed by the telomeric repeat amplification protocol (TRAP) and the presence of telomerase compared with the histological diagnosis and the proliferation index. RESULTS: A high degree of positivity for telomerase activity was found in glioblastomas and atypical/anaplastic meningiomas. Telomerase activity was poorly detected in anaplastic astrocytomas. CONCLUSIONS: The TRAP assay seems to be a valuable index for identifying meningeal tumours with aggressive behaviour.     

Function of HSP90 and p23 in the telomerase complex of thyroid tumors

Recently, studies on endocrine tumors revealed a potential role of telomerase in the dedifferentiation and/or malignant transition of these tumors. Telomerase is a ribonucleoprotein complex that includes the telomerase RNA component (hTR), the telomerase-associated protein (TP1), and the telomerase catalytic subunit (hTERT). Previously, the chaperones p23 and HSP90 have been described as additional telomerase regulators. To test whether the interactions of these genes are reflected in the dedifferentiation of thyroid tumors, we determined their mRNA and/or protein expression in 30 normal (tumor-free) thyroid tissues (NT), 35 follicular adenomas (FAD), 42 papillary carcinomas (PTC), 38 follicular carcinomas (FTC), 25 poorly differentiated carcinomas (PDTC), and 34 undifferentiated carcinomas (UTC). We then compared the results with telomerase activity. RT-PCR analysis revealed that TP1 was ubiquitously expressed. hTR was found in 50-94% of malignant tumors, in contrast to 7% of NT and 26% of FAD. hTERT was clearly associated with aggressive biological behavior. Ninety-two to 100% of the malignant tumors were positive for hTERT protein, whereas NT and FAD were negative in 100% and 94%, respectively. HSP90 mRNA and protein showed a close relationship to hTERT. p23 protein was negative in NT and positive in 3% of FAD, 39% of FTC, 40% of PTC, 44% of PDTC and 47% of UTC. High telomerase activity was measurable in hTERT and HSP90-positive tissues only. Our data show that the common expression of hTERT and HSP90 regulates telomerase activity in thyroid carcinomas. Chaperone p23 is involved in the telomeric complex to a lesser extent, but its expression is stronger in carcinomas than in non-malignant thyroid tissues. The expression profile of telomerase components represents an additional prognostic marker that may identify more aggressive thyroid tumors.     

Telomerase activity in melanocytic lesions: A potential marker of tumor biology

Telomerase activation, being a cardinal requirement for immortalization, is a crucial step in the development of malignancy. With a view toward diagnostic and biological aspects in melanocytic neoplasia, we investigated the relative levels of telomerase activity in 72 nevi and 16 malignant melanomas by means of a modified telomeric repeat amplification protocol (TRAP) assay, including an internal amplification standard. We further compared telomerase activity with the expression of two different proliferation-specific proteins, Ki-67 and repp86, a protein expressed exclusively in the cell cycle phases S, G2, and M. Telomerase activity was associated with the overall growth fraction (Ki-67) but showed a closer correlation with the expression of repp86. Both telomerase activity and proliferation indices discriminated clearly between malignant melanomas and nevi, but not between common and dysplastic nevi. Nonetheless, a portion of nevi exhibited markedly elevated telomerase activity levels without proportionally increased proliferation. This was independent of discernible morphological changes. Clinicopathological correlations showed an association between high telomerase activity and early metastatic spread in melanomas, linking telomerase to tumor biology. Our results provide arguments in favor of an occasional progression from nevi to melanomas and imply that proliferation measurements in combination with telomerase assays may help to elicit early malignant transformation that is undetectable by conventional morphology.     

Clinical implications of telomerase activity in resected hepatocellular carcinoma.

This study investigated the relationship of telomerase activity to its clinical implications in hepatocellular carcinoma (HCC). Telomerase activity was quantatively examined by a non-radioisotope quantitative system based on a TRAP-eze (ONCOR) in 42 HCC nodules and 40 non-cancerous liver tissue adjacent to the HCC nodules, obtained by surgical resection. Telomerase activity was confirmed in 37 HCC nodules (88.1%), being more statistically intense in de-differentiated tumors (p<0.01). Telomerase activity was positive in 9 cases (22.5%) of non-cancerous liver tissue and its activation was closely associated with the biologically malignant potential of the tumor itself such as the histological grade, portal vascular invasion, and intrahepatic metastasis (p<0. 05). The disease-free survival rate with its activity was also statistically lower than that without its activity of the non-cancerous tissue (p<0.01). These results indicate that telomerase may be a useful marker of biological characteristics in HCC and may lead to more effective surgical procedures.     

Telomerase activity in well-differentiated papillary thyroid carcinoma corrrelates with advanced clinical stage of the disease

Clinical relevance and stage correlation of telomerase activity in well-differentiated papillary thyroid carcinoma (WD PTC) has not been well determined, as its reported activity could be due to the analysis of tumors with lymphocytic infiltrates or aggressive variants of papillary carcinomas. We conducted a prospective study of telomerase activity in WD PTC without inflammatory infiltrates and correlated it with clinical stage. Fifty WD PTCs were analyzed for telomerase activity by PCR-based TRAP (telomeric repeat amplification protocol) assay. Results were correlated with stage and other clinicopathologic variables. Twenty-one (42%) WD PTCs demonstrated telomerase activity. The enzyme was detected more frequently in stage III/IVa WD PTCs (p=0.02) and in tumors with extra thyroidal extension (p=0.04). The risk of presenting advanced disease (stage III/IVa) and extrathyridal growth was significantly increased in telomerase-positive tumors (p=0.01; odds ratio [OR] 4.4 [95%Cl 1.3–14.7]) and (p=0.04; OR 3.6 [95%Cl 1.1–11.7]), respectively. Also, a correlation was found between telomerase activity and age. There was no correlation of telomerase activity with gender, histologic variant, tumor size, or cervical lymph node metastasis. Telomerase activity was observed in 42% of WD PTC and was detected more frequently in AJCC TNM stage III/IVa cases. This finding suggests that telomerase deregulation could be involved in tumor progression.     

Clinical usefulness of telomerase assay for the detection of lymph node metastasis in patients with oral malignancy

OBJECTIVE: Telomerase is considered a diagnostic marker of malignancy. We investigated the usefulness of telomerase assay for the detection of lymph node micrometastasis. METHODS: Sixteen cervical lymph nodes with metastasis of oral cancer and 20 benign lymph nodes were studied. The oral cancer cell line was used to estimate the sensitivity for telomerase assay. Telomerase activity was measured by semiquantitative telomeric repeat amplification protocol. RESULTS: There was a significant difference between malignant and benign lymph nodes. The telomerase activity of 50 mg of lymph nodes with 103 or more cancer cells differed from that of control lymph nodes. Lymph nodes with 102 or fewer tumor cells expressed similar levels as benign lymph nodes. CONCLUSIONS: In addition to routine histologic examination, telomerase assay is considered a useful tool for the detection of lymph node metastasis in patients with oral malignancy.     

Change of telomerase activity in rectal cancer with chemoradiation therapy

Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant cells and is inactive in most normal somatic cells, with the exception of proliferative stem cells, germ cells and activated lymphocytes. Measuring telomerase activity clinically may provide useful diagnostic and prognostic information of cancer. The purpose of this study was to investigate the change in telomerase activity following chemoradiation in rectal cancer, which almost always produces positive enzymatic activity. A total of 24 tumor tissue samples were used in this study, consisting of 12 paired specimens before and 4 weeks after chemoradiation. Telomerase activity was determined by PCR-based telomeric repeat amplification protocol (TRAP) assay. The telomerase activity was positive in 10 out of 12 patients (83%) in pre-irradiated and post-irradiated states. The levels of telomerase activity was decreased in 8 out of 10 patients after chemoradiation (80%) and two cases showed no change in enzymatic activity. One case showed no activity in either sample. The other case showed no enzymatic activity in the pre-irradiated sample, but showed weak activity in the post-irradiated sample. These data indicate that telomerase activity in rectal cancer is reduced after neoadjuvant chemoradiation therapy, possibly suggesting a mechanism of downstaging following chemoradiation therapy in cancer.     

反义hTERT/PTEN联合基因治疗恶性胶质瘤的体内外研究

目的探讨腺病毒介导的反义hTERT和野生型PTEN联合基因转染对恶性胶质瘤细胞生长的影响。方法用细菌内高效同源重组系统构建的含有hTERT反义序列及野生型PTEN的腺病毒在体内外单独或联合转染恶性胶质瘤细胞系U251，检测肿瘤细胞生长情况、端粒酶活性、蛋白表达、细胞周期变化等。结果在体外试验中单独或联合转染都能明显抑制肿瘤细胞的生长，以联合转染效果最明显，转染后第6天联合转染组的细胞生存率为37．6％，端粒酶活性水平和hTERT蛋白表达水平明显下降，分别为28．8TPG、0．2106，PIEN蛋白表达水平上升为0．9630；在体内试验中肿瘤生长也明显减慢。结论腺病毒介导的反义hTERT和野生型PTEN联合转染能明显抑制恶性胶质瘤细胞的生长。     

卵巢肿瘤组织中端粒酶活性的表达

目的分析端粒酶活性在卵巢肿瘤中的表达,探讨端粒酶活性作为卵巢肿瘤诊断肿瘤标记物的意义。方法取冰冻肿瘤组织,采用端粒重复序列扩增法（Telomeric Repeat Amplication Protocol）,结合银染,共测定了27例卵巢癌、5例良性卵巢肿瘤、22例卵巢癌癌旁组织以及12例正常卵巢组织中端粒酶活性,并分析其与组织分级、FIGO分期、病理类型以及有无转移的关系。PCR产物以采用聚丙烯酰胺凝胶电泳分析,以出现特征性的6-bp间隔的特征性阶梯状条带为阳性。出现〉100bp的条带为强阳性,出现〈99bp的条带为中低阳性。结果本实验共对66份标本进行了检测,12例正常卵巢组织中,仅有2例表达端粒酶活性;5例卵巢良性肿瘤中,1例表达端粒酶弱阳性。27例卵巢恶性肿瘤标本中,有23例表达端粒酶活性（其中强阳性16例）;在检测的22例卵巢上皮癌的癌旁组织中,有12例表达端粒酶弱阳性。卵巢恶性肿瘤的端粒酶活性表达阳性率显著高于卵巢良性肿瘤（P=0.0090）和正常卵巢（P=0.0000）。Ⅰ-Ⅱ期患者端粒酶活性阳性率71%,Ⅲ-Ⅳ期患者端粒酶活性阳性率90%（P=0.269）。而强阳性率Ⅲ-Ⅳ期明显高于Ⅰ-Ⅱ期（P=0.009）。在肿瘤类型和组织学分级中,端粒酶阳性表达率均没有显著差异。结论在卵巢恶性肿瘤组织中端粒酶活性有较高的表达率;早期和晚期卵巢肿瘤组织中端粒酶活性阳性率没有明显差异,但晚期卵巢肿瘤组织中的端粒酶活性程度较高;端粒酶活性有可能作为早期诊断卵巢肿瘤的标记物之一,并可以作为卵巢肿瘤判断预后的指标。     

Divergent patterns of telomere maintenance mechanisms among human sarcomas: sharply contrasting prevalence of the alternative lengthening of telomeres mechanism in Ewing's sarcomas and osteosarcomas

Two types of telomere maintenance mechanisms (TMMs) have been described in human tumors: telomerase activation and alternative lengthening of telomeres (ALT). Although the vast majority of epithelial tumors rely on telomerase activation, many mesenchymal tumors rely on ALT for telomere maintenance, but within this tumor group, the TMMs used by translocation-associated sarcomas have not been systematically studied. We studied telomere lengths and telomerase expression and activity in 30 uncultured tumor samples and in 10 cell lines of Ewing's sarcoma, a prototypical translocation-associated sarcoma, and compared the data to an identical analysis of 60 osteosarcomas, the most common type of sarcoma lacking a specific translocation. Telomerase activity was demonstrated in 21 Ewing's sarcoma tumor samples (70%) and in 9 of 10 Ewing's sarcoma cell lines. Evidence of ALT, indicated by the presence of long and heterogeneous telomeres, was observed only in the cell line without telomerase activity and in none of the 30 Ewing's sarcoma tumor samples. The 9 Ewing's sarcoma patients whose tumors lacked detectable telomerase activity did not differ significantly from the remaining patients in age, stage, EWSR1-FLI1 fusion type, prevalence of TP53 point mutations, or overall survival. The low prevalence of ALT in Ewing's sarcoma contrasted sharply with our data on TMMs in 60 osteosarcomas, which showed ALT in 38 of 60 cases (P<0.0001). The present study, together with emerging published data on other sarcoma types, suggests that a predominance of telomerase activation in the absence of ALT may characterize sarcomas with specific chromosomal translocations (such as Ewing's sarcoma), whereas a high prevalence of ALT appears typical of sarcomas with nonspecific complex karyotypes (such as osteosarcoma).