Flow cytometric study of lymphocytes associated with thymoma and other thymic tumors

BACKGROUND. A large number of immature T lymphocytes in thymoma may reflect the biological function of the neoplastic epithelial cells. However, to confirm that this lymphocyte-inducing activity is unique to thymoma, lymphocytes associated with other thymic tumors need to be studied. MATERIALS AND METHODS. We used flow cytometry to study lymphocytes recovered from various thymic tumors (65 thymomas, 24 with myasthenia gravis; 5 thymic cancers; 5 germ cell tumors including 3 needle biopsy samples; and 2 other tumors) and results were analyzed in reference to those from 36 normal thymuses. RESULTS. The frequency of CD4(+)CD8(+) (DP) thymocytes in the normal thymus declined with age (0.9-94%, r = -0.83, P < 0.001) reflecting the physiological involution. Association of lymphocytes with this DP phenotype was unique to thymoma: 61 of 65 thymomas but none of the other thymic tumors had more than 3% DP cells (frequency of DP cells; thymoma without MG, 59.5 +/- 31.4%; thymoma with MG, 59.4 +/- 22.1%; and other thymic tumors, 0.8 +/- 1.0; mean +/- SD). All the thymic tumors associated with myasthenia gravis were thymomas and had more than 18% DP cells. CONCLUSION. The presence of DP cells in thymomas but not in other tumors suggests that DP cells are induced by the epithelial cells of thymoma. This characteristic may help diagnose thymic tumors; the presence of more than 3% DP cells suggests a thymoma. Also, association of myasthenia gravis suggests a thymoma.     

Telomerase activity in human renal cell carcinoma

PURPOSE: Telomerase activity has been detected in a wide variety of human tumors. The present study evaluated telomerase activity in association with the acquisition of renal cell carcinoma (RCC). METHODS: Telomerase activity was examined in 30 RCC and the adjacent normal kidney tissue, obtained as surgical specimens. The activity was assayed by polymerase chain reaction-based telomeric repeat amplification protocol assay. RESULTS: Among the 30 RCC, 18 (60%) displayed telomerase activity, whereas none of the normal tissue samples exhibited it. Subdivision of the tumors according to telomerase activity did not reveal any obvious difference in distribution with regard to tumor size, stage, histocytological subtype, or DNA-ploidy. However, a statistically significant relationship was found between the frequency of telomerase-positive activity and both serum immunosuppressive acidic protein level in the patient and tumor grade (P<0.05). There was no significant difference in the recurrent-free survival and the disease-specific survival between patients with positive telomerase activity and patients with negative activity. CONCLUSION: The present results indicate that telomerase activity might be related to the progression of RCC and thus a marker of malignant potential.     

Telomerase activity in renal cell carcinoma by modified telomeric repeat amplification protocol assay

BACKGROUND: Previous assessments by the conventional telomeric repeat amplification protocol have not been reliable for the quantitation of telomerase activity. We, therefore, determined telomerase activity in renal cell carcinoma (RCC) tissue by the modified sensitive telomeric repeat amplification protocol assay. METHODS: Telomerase activity was examined in 23 cases of RCC and in the adjacent normal kidney tissue, and assessed for associations with clinical and pathological variables of the disease. RESULTS: The linearity and quantitation of the modified method was confirmed. Mean telomerase activity of RCC (1987.889 +/- 1232.801 units) was significantly greater than that of normal renal tissue (173.467 +/- 241.893 units) (P = 0.0001). Telomerase activity in RCC was, however, not associated with clinical or pathological variables such as clinical stage (P = 0.8941), grade (P = 0.8043) or pathological subtype (P = 0.9739). CONCLUSION: The results suggest that telomerase might play a crucial role in an initial step of the development of RCC, but not in the progression of the disease.     

Telomerase activity is correlated with lower grade and lower stage bladder carcinomas

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that compensates for the progressive erosion of telomeres. The increasing interest in telomerase is motivated by the demonstration that most human carcinomas are telomerase positive. The potential use of telomerase activity in bladder carcinomas using a urine sample has been reported in several studies. However, little is known about the detection of telomerase activity in bladder carcinoma tissues. Herein, we investigate telomerase activity in bladder carcinoma tissues according to grade (G) and stage. MATERIAL AND METHODS: Telomerase activity was assayed by polymerase chain reaction enzyme-linked immunosorbent assay methods. Malignant lesions were assessed in 37 patients with bladder carcinoma and no malignant lesions were assessed in five patients with dysplasia or inflammatory bladder lesions. RESULTS: Twenty-three out of 37 carcinoma samples were telomerase-positive and one out of five control samples without carcinoma was telomerase-positive. The positive rates according to stage and grade were 83.3% for superficial and 42.1% for invasive stages and 83.3% for G1, 66.7% for G2 and 40.0% for G3. Telomerase activity was correlated with lower grade and lower stage bladder carcinomas. CONCLUSION: These results strongly suggest that reactivation of telomerase may differ between superficial and invasive bladder carcinomas and also between low grade and high grade bladder carcinomas.     

Telomerase as an independent prognostic factor in head and neck squamous cell carcinoma

BACKGROUND: Telomerase activity has been found to be associated with many cancers, including head and neck squamous cell carcinoma (HNSCC). We examined the association of telomerase activity with the clinical outcome of patients with HNSCC. METHODS: A PCR-based enzyme immunoassay method was used to measure telomerase activity in 217 matched (grossly normal and cancerous) tissues from patients with HNSCC. Pearson chi-square test was used to analyze the correlation of telomerase activity with clinicopathologic parameters. Kaplan-Meier method and Cox logistic regression model were used for prognostic analysis. RESULTS: Of the 217 tissues assayed, 4.1% of the normal and 63.3% of the cancer tissues had high levels of telomerase activity. Telomerase activity was shown to be statistically correlated with extracapsule spreading (ECS) of lymph nodes (p =.005) and overall survival (p =.003). On multivariant analysis, overall stage (p =.007), tumor depth (p =.045), and telomerase activity (p =.008) showed independent variables associated with poor survival. CONCLUSIONS: Telomerase activity has been shown to be an independent prognostic factor for survival in cases of HNSCC. Telomerase may be a potential molecular target for clinical use in prognostication and therapy in cases of the disease.     

Flow cytometric analysis of lymphoid cells in thymic epithelial neoplasms.

OBJECTIVE: There have been conflicts concerning the criteria for diagnosing malignant epithelial neoplasms of thymic origin. To differentiate thymic carcinomas from thymomas, the maturation stage of T-lineage lymphoid cells infiltrating thymomas and thymic carcinomas was examined by flow cytometry to associate it with the degree of tumor malignancy. METHODS: Multidimensional flow cytometric analysis was performed on the lymphoid cells extracted from 27 thymic epithelial neoplasms (14 encapsulated thymomas, ten invasive thymomas, and three thymic carcinomas) by using anti-CD3, -CD4, -CD8, -CD10, -CD20, -CD38, -CD45RA, and -CD45RO monoclonal antibodies. RESULTS: CD4 and CD8 were co-expressed on 76.8% of the lymphoid cells in encapsulated thymoma (N=14), 59.2% in invasive thymoma (N=10), and 6.7% in thymic cancer (N=3). The percentage of CD4- or CD8- single positive cells was 11.4% in encapsulated thymoma, 23.9% in invasive thymoma, and 77.7% in thymic cancer. The percentage of CD10-positive cells was 20.8% in encapsulated thymoma, 13.2% in invasive thymoma, and 6.0% in thymic cancer. The percentage of CD20-positive cell was 2.6% in encapsulated thymoma, 3.3% in invasive thymoma, and 31.6% in thymic cancer. There were significant statistical differences in the percentages of CD4/CD8 double positive cells, CD4- or CD8-single positive cells, CD10-positive cells and CD20-positive cells among the three groups. Two cases classified as invasive thymoma by pathohistological examination, however, showed the infiltration of mature lymphocytes like as thymic cancers. CONCLUSIONS: CD4+CD8+ or CD10+ T-lineage cells were the most reliable markers of the benignancy of thymic epithelial tumors. CD4- or CD8-single positive cells or CD20-positive cells were characteristic in thymic carcinoma. Flow cytometry on the maturity of lymphoid cells infiltrating thymic epithelial tumors was feasible for determining their degree of malignancy. Some invasive thymomas showed the intermediate characteristics with thymomatous epithelia and mature lymphoid cells.     

Prognostic impact of telomerase activity in non-small cell lung cancers

OBJECTIVE: To evaluate the clinical significance of telomerase activity, particularly in terms of prognostic impact, in non-small cell lung cancer (NSCLC). SUMMARY BACKGROUND DATA: Telomerase activity has been found in various tissues. The activation of telomerase is considered necessary for the immortalization of human tumor cells, including NSCLC. METHODS: The authors studied 103 NSCLC specimens using a polymerase chain reaction based on a telomeric repeat amplification protocol assay. RESULTS: Telomerase activity was detected in 85 (82.5%) of 103 NSCLC specimens but in none of the paired normal lung tissue specimens. More cases of positive telomerase activity were observed in the group with advanced disease and in the group with poorly differentiated tumors. Such factors as the mean age at surgery, sex, smoking, histologic type, and size of tumor extension did not correlate with the telomerase activity. The Kaplan-Meier survival curves in all patients with NSCLC demonstrated that patients with telomerase-positive tumors survived for a significantly shorter period than those with a telomerase-negative tumor (p = 0.0058). According to a multivariate analysis, telomerase activity was identified as an independent prognostic factor (RR = 8.62, p = 0.035). CONCLUSIONS: Telomerase activity was one of the most important prognostic factors in patients with NSCLC, and its potential prognostic implication was independent of tumor stage.     

Consistent decrease in telomere length in parathyroid tumors but alteration in telomerase activity limited to malignancies: preliminary report

Telomerase is known to be activated and telomere length altered in various types of malignant and benign tumors, but whether this is also the case for parathyroid lesions has hitherto been unclear. We therefore investigated telomerase activity and telomere length in 3 parathyroid metastatic cancers, 6 adenomas, 2 cases of parathyroid hyperplasia, and 16 samples of normal parathyroid tissue. Telomerase activity, assayed by the telomeric repeat amplification protocol, was detected in all of the parathyroid cancers (100%), in none of the 8 parathyroid benign lesions, and in only 1 of the 16 normal parathyroid samples (8.3%). Telomere length, determined by the terminal restriction fragment assay, was reduced in the tumor tissues with a mean telomere length of 8.23 +/- 0.86 kbp compared with the 12.61 +/- 0.81 kbp for the 16 age-matched subjects (p = 0.002). The results indicate that telomerase activity and telomere length may reflect the biologic behavior of individual parathyroid lesions.     

The association between telomerase, histopathological parameters, and KI-67 expression in breast cancer.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme that appears to play an important role in carcinogenesis. Telomerase reactivation seems to be associated with immortalization and malignancy. METHODS: Using a polymerase chain reaction (PCR)-based assay known as the TRAP (telomeric repeat and amplification protocol) assay, we examined telomerase activity in 60 breast specimens prospectively collected from 39 patients undergoing elective breast surgery in our center. The specimens included adjacent noncancerous breast (n = 21), benign breast disease (n = 5), and infiltrating carcinoma (n = 34). Ki-67 expression was determined in 32 invasive breast cancer specimens using immunohistochemistry techniques. The histopathological features were determined by light microscopy by an experienced breast pathologist. RESULTS: Telomerase activity was detected in 24 (71%) of 34 infiltrating carcinomas. None of the adjacent noncancerous specimens nor the benign breast lesions expressed telomerase activity. Telomerase reactivation was significantly associated with nodal metastasis and Ki-67 expression. There was no significant association between telomerase activity and menopausal status, tumor grade, or tumor size. CONCLUSIONS: Telomerase reactivation is associated with the acquisition of malignancy in the human breast. Telomerase activity is significantly associated with nodal metastasis and cellular proliferation as measured by Ki-67 expression in human breast cancer.     

Telomerase Activity in Tumor and Remnant Liver as Predictor of Recurrence and Survival in Hepatocellular Carcinoma after Resection

Background and Aim Results after curative liver resection in hepatocellular carcinoma are unsatisfactory with regard to high postoperative intrahepatic recurrence and liver failure. This study evaluates telomerase activity in liver with and without tumor as a predictor of recurrence and survival. Materials and Methods Liver tissue with and without tumor from 53 hepatocellular carcinoma patients receiving curative resection during the period of 1998–2000 was used for detecting telomerase activity by PCR-ELISA. Clinicopathological data were compared to identify predictors of recurrence and survival. Results Telomerase activity was detected in 98% of liver tissue with tumor and 70% liver tissue without. Telomerase activity in cancerous liver correlated significantly with HCV infection (P = 0.012) and cirrhotic change in liver parenchyma (P = 0.006). Telomerase activity in non-cancerous liver correlated with high serum AFP level (P = 0.002). The telomerase activity of liver tissue with and without tumor is significant higher in patients with recurrence than in those without recurrence, 413.7 ± 100.5 versus 110.8 ± 32.7, P = 0.006, and 34.7 ± 14.2 versus 4.2 ± 1.4, P = 0.039. Recurrence could be predicted by abnormally high tumor telomerase activity (P = 0.026) or by advanced TNM stage (P = 0.001). TNM stage or high serum ALT level could predict multinodular intrahepatic recurrence (P = 0.028 and P = 0.030). High serum AFP combined with high telomerase activity in liver without tumor had a significant ability to predict poor survival (OR: 11.19, CI: 1.95–64.12, P = 0.007). Conclusion Tumor telomerase is an independent predictor of recurrence. Simultaneous high remnant liver telomerase and high serum AFP is a strong negative predictor of survival.     

Telomerase activity in the human breast

Telomerase is a ribonucleoprotein enzyme which appears to play an important role in carcinogenesis. Telomerase reactivation seems to be associated with immortalization and malignancy. Using a PCR-based assay, we examined telomerase activity in 50 breast tissue specimens, prospectively obtained from 37 women undergoing elective breast surgery. The specimens examined included normal breast (n=13), benign breast lesions (n=5), ductal carcinoma in situ (n=8) and infiltrating ductal carcinoma (n=24). All normal breast, benign breast and DCIS specimens lacked telomerase activity. Sixteen (67%) of 24 infiltrating carcinomas. In infiltrating ductal cancer, there was a statistically significant association between telomerase activity and nodal metastasis. The present results indicate that telomerase activity is associated with acquisition of invasive malignancy in the human breast and may have a role in complementing cytopathological diagnosis. Telomerase activity as a prognostic marker should be included in future validation studies. In DCIS, telomerase activity may be a late event associated with invasion of the basement membrane.     

Azidothymidine induces apoptosis and inhibits cell growth and telomerase activity of human parathyroid cancer cells in culture.

Telomerase activity has been correlated to parathyroid carcinoma. Because its role in acquisition of a malignant phenotype by parathyroid cells is unclear, we treated telomerase-positive cultured human parathyroid cancer cells with the telomerase inhibitor AZT, evaluating cell telomerase activity, cytotoxic effects, growth, and morphological changes. In vitro exposure of these cells to AZT correlated with inhibition of cell proliferation. INTRODUCTION: Parathyroid carcinoma represents an uncommon cause of primary hyperparathyroidism, whose spectrum of clinical presentation, degree of malignancy, and prognosis are difficult to be properly identified. Neck surgery, specifically an en bloc resection of primary tumor, is the only curative treatment. Alternatively, affected patients could undergo repetitive palliative surgical exeresis of metastatic nodules. It has been previously shown that telomerase activity is specifically present in parathyroid carcinoma cells, being absent in hyperplastic and adenomatous tissues. Thus, determination of telomerase activity could represent either a useful diagnostic molecular marker for human parathyroid carcinoma or a potential target for pharmacological intervention in a malignant neoplasia usually resistant to chemo- and radiotherapeutic interventions. MATERIALS AND METHODS: To further investigate the role of telomerase activity in acquisition of a malignant phenotype by parathyroid cells, we treated telomeric repeat amplification protocol-positive cultured human parathyroid cells with the telomerase inhibitor zidovudine, 3'-azido-3'deoxythymidine (AZT), evaluating cell telomerase activity, growth characteristics, potential cytotoxic effects, and morphological changes. RESULTS: Our findings indicate that in vitro exposure of human parathyroid cancer cells to AZT resulted in intracellular accumulation of AZT-monophosphate (AZT-MP) and inhibition of telomerase, which correlate with inhibition of human parathyroid cancer cell proliferation. Moreover, we also found that AZT induced an apoptotic rather than a necrotic type of cellular death. None of these effects were observed in human adenomatous parathyroid cells in culture. CONCLUSIONS: Altogether these results indicate that AZT may be a highly effective agent against cancer parathyroid cells proliferation, which is an extremely important observation for a neoplasia which shows lack of response to classical pharmacological and physical antiblastic treatments.     

A quantitative assay for telomerase enzyme activity predicts severity of disease in neuroblastoma

PurposeTo determine whether telomerase enzyme activity correlates with tumor stage, grade, and prognosis using a quantitative telomerase assay.MethodA new semiquantitative assay for telomerase enzyme activity (Telomerase Repeat Amplification Protocol–enzyme-linked immunosorbent assay) was employed on 21 neuroblastoma specimens. Enzyme-linked immunosorbent assay results were compared to tumor stage, patient age, and overall tumor aggressiveness. The principle investigator was blinded to all clinical data. A Student’s t test was used to determine significance (p < 0.05).ResultsTelomerase activity correlated with stage of tumor (p < 0.007). Age of patient did not correlate with telomerase activity independently.ConclusionsTelomerase enzyme activity was predictive for neuroblastoma stage. This implies that increased telomerase activity is involved in tumor dedifferentiation. The semiquantitative telomerase assay may be used as a rapid prognostic indicator and serve as a guide to initiating treatment in childhood neuroblastoma.     

Telomerase activity in malignant and benign renal tumors

OBJECTIVES: An important characteristic of malignant cells is their unlimited replicative potential, their immortality. In conferring this immortality, the enzyme telomerase is believed to play a crucial role. The detection of telomerase activity provides new knowledge regarding the biologic growth behavior of tumors and offers new diagnostic and therapeutic tools. METHODS: In the present study the sensitive TRAP assay (telomeric repeat amplification protocol) was used to examine 44 malignant renal tumors and 8 benign tumors of the kidney and 52 specimens of normal renal tissue for telomerase activity. RESULTS: Telomerase activity was detected in 63% of tissue samples obtained from histologically confirmed renal cell carcinomas. In cases of renal cell carcinoma restricted to the kidney, telomerase activity was detected in 58%. In cases in which tumor growth has progressed beyond the limits of the organ, telomerase activity was found in 69%. This stage dependence, however, did not reach statistical significance. No correlation to tumor grading was observed. Telomerase activity was found less frequent in chromophobe renal cell carcinomas. Neither the 8 benign renal tumors (4 oncocytomas and 4 angiomyolipomas) nor the specimens of normal kidney showed any evidence of telomerase activity. CONCLUSIONS: The proportion of remarkable slow-growing renal cell carcinomas showing telomerase activity is less than in other malignancies and may correlate with biologic growth behavior. Possible explanations include the presence of an alternative pathway, called ALT (alternative lengthening of telomeres) and an association with the loss or presence of the telomerase suppressor on the short arm of chromosome 3. Prolonged follow-up will be of special interest to determine whether lack of telomerase activity predicts favorable outcome.     

Telomerase activity in periampullary tumors correlates with aggressive malignancy

OBJECTIVE: To determine the presence of telomerase activity in a variety of periampullary malignancies and pancreatic diseases and quantify its activity to establish any association with the stage or aggressiveness of malignancy. SUMMARY BACKGROUND DATA: Progressive shortening of telomeres, repetitive DNA sequences at the ends of chromosomes, plays a role in cell senescence. Telomerase catalyzes conservation of telomeric repeats and may promote cell immortality and hence malignancy. It is absent in normal tissues but upregulated in more than 80% of cancers. METHODS: Fresh specimens of 62 periampullary tumors were snap-frozen in liquid nitrogen and adjacent tissue was formalin-fixed for histopathology. The telomerase repeat amplification protocol (TRAP) was used to obtain telomerase DNA products. These were separated with gel electrophoresis, stained with SYBR green, and quantified by densitometry. Findings were confirmed with a fluorometric TRAP assay in which fluorescent primers specific for telomerase were selectively amplified in its presence. RESULTS: Telomerase activity was upregulated in 26 of 33 periampullary malignancies (79%): 17 of 21 pancreatic adenocarcinomas (81%), 2 of 2 cholangiocarcinomas, 2 of 2 duodenal carcinomas, and 5 of 8 ampullary carcinomas (63%). Poorly differentiated periampullary tumors had significantly higher telomerase activity than well-differentiated tumors, and tumors larger than 2 cm had significantly higher telomerase activity than those 2 cm or smaller. Pancreatic ductal adenocarcinomas with lymph node metastases had significantly greater activity than node-negative cancers. Two of 11 intraductal papillary mucinous tumors were positive for telomerase activity, but only in foci of invasive carcinoma. Chronic pancreatitis (n = 7), serous cystadenomas (n = 5), benign mucinous cystic neoplasms (n = 4), neuroendocrine cancer (n = 1), and acinar cell carcinoma (n = 1) had no detectable telomerase activity. CONCLUSION: Telomerase activity is common in periampullary carcinomas. The magnitude of activity correlates with aggressiveness in pancreatic adenocarcinoma and may prove useful as a molecular index for biologic staging.     

PCR-ELISA法检测膀胱肿瘤患者尿脱落细胞端粒酶活性

目的：探讨尿脱落细胞端粒酶活性变化在膀胱肿瘤诊断中的作用。方法应用PCR－ELISA法检测53例膀胱肿瘤患者尿液脱落细胞端粒酶的活性。结果：非膀胱肿瘤和膀胱肿瘤患者尿液脱落细胞端粒酶活性阳性率分别为64．15％（34．53）和7．69％（2／26）,健康对照者7例均为阴性,膀胱肿瘤患者与正常人及非膀胱肿瘤患者的端粒酶活性分别相比,差别均有极显著性意义（P＜0．001）。但端粒酶活性与肿瘤的分期分级无相关性。结论：尿脱落细胞端粒酶活性检测可以作为诊断膀胱肿瘤的无创性检测方法,但不能预测膀胱肿瘤的临床分期分级。     

Clinical and pathologic predictors of survival in patients with thymic tumors

BACKGROUND: The aim of this study is to evaluate the impact of thymectomy in patients with thymic neoplasms and to identify clinical and histopathological factors associated with improved long-term outcome of surgery. METHODS: We treated 74 patients between February 1987 and July 1993. There were 29 total and 36 simple thymectomies. These last cases, all non-myasthenic, had benign thymomas (n=30) but 6 had thymic carcinomas. Nine tumors were no-resected (5 thymomas and 4 thymic carcinomas). Minimum follow-up by Department of Thoracic Surgery Istituto Nazionale Tumori was 60 months after thymectomy. We divided the specimens according to Marino and Muller-Hermelink's classification: 54 thymomas, 18 thymic carcinomas and 2 no-diagnosis specify thymomas. There were 53 stage I, 1 stage II, 13 stage III, 5 stage IVa and 2 stage IVb according to Masaoka. RESULTS: Forty-six patients with treated thymoma were alive without disease at the end of follow-up, the remaining 8 died from recurrence in 6, a new tumor in 1 and a heart attack in the last. Of 18 thymic carcinomas 9 were alive at the end of follow-up (1 with recurrence), only 4 dead from recurrence. The actuarial survival of patients with thymomas was 88.5% at 5 years, (73.6% in cortical type, 85.7% in medullary type, 93.9% in mixed type, 100% in predominantly cortical type). Myasthenia gravis didn't influence the survival: 87.3 (no MG) vs 90%. Advanced stage thymomas significantly increased the risk of death from early stage I: 32.4 vs 100% at 5 years. In thymic carcinoma patients with well-differentiated thymic carcinoma (WDTC) died less than others: the actuarial probability of survival at 5 years was 90 vs 68%. CONCLUSIONS: Thymectomy was the best treatment to long term outcome. In our experience, survival was related to histotype and to local extension of tumor.