Pitfalls in TRAP assay in routine detection of malignancy in effusions

Telomerase has been found to be reactivated in a majority of cancers but is inactive in most somatic cells. Our principal goal was to determine the potential use of the telomeric repeat amplification protocol (TRAP) assay as marker for malignancy in cytological effusions. The simple selection criterion was the cytological diagnosis, and routine samples were classified into malignant (58 samples) and nonmalignant (233 samples). Of the malignant samples, 44/58 (76%) were positive by TRAP assay. Of the 14 telomerase-negative cytology-positive samples, RNA integrity was poor in 9, indicating suboptimal sample conservation for molecular analysis. In 3 of the remaining 5 samples with a negative TRAP assay, a high number of malignant cells was observed, and these cells might have been telomerase-negative. Thus, the sensitivity of TRAP assay for the presence of malignant cells was about 76%. In the cytologically nonmalignant effusions, the presence of telomerase activity was observed in 24% (55/233). Of these, 6% were highly suspicious for malignancy, 9% were doubtful, and 9% were cytologically nonmalignant effusions confirmed by a follow-up of 12 mo or more. According to these data, the specificity of the TRAP assay to detect tumor cells in effusions ranged only between 82-91%. Our results indicate that, although the TRAP assay is positive in 6-15% of putative malignant effusions, the relatively high number of TRAP false-negative and false-positive cases renders this test unsuitable for routine diagnostic purposes.     

Quantitative detection of telomerase activity by real-time TRAP assay in the body fluids of cancer patients

Real-time TRAP assay was developed to improve the sensitivity and quantitative detection of telomerase activity in the body fluids of cancer patients. The sensitivity and clinical significance of the real-time TRAP assay was investigated. Real-time PCR protocol was modified by using ACX primer and SYBR green mixture from the process of TS primer extension. Real-time TRAP showed high correlation (r2=0.843, p=0.001) and sensitivity (25 times higher) compared to conventional TRAP. Of 164 samples, there were 8 positives in cytology (4.9%), 7 (4.3%) using the conventional TRAP, and 41 (25%) using real-time TRAP. In cytology positive samples, real-time TRAP showed a higher positivity than conventional TRAP (75% vs 63%) suggesting a higher sensitivity in the body fluids. There was a tendency towards a longer progression-free duration in telomerase negative patients than in positive patients, as determined by conventional and real-time TRAP. The diagnostic interval between the first positivity documentation and clinical progression was short in the order of real-time TRAP, conventional TRAP and cytology. In conclusion, real-time TRAP assay can detect telomerase activity at an earlier phase of cancer progression and can replace conventional TRAP assay for detecting the telomerase activity in body fluids.     

In situ telomerase activity in pleural effusions: a promising marker for malignancy

Telomerase activity in 16 pleural effusions was studied using an in situ telomerase repeat amplification protocol (TRAP) assay on cytospin preparations. Six of nine cytologically malignant specimens contained telomerase-positive cells (67%), and in two further specimens, suspicious positive cells were seen. Two of four atypical specimens contained telomerase-positive cells, whereas two benign cases were telomerase-negative. No mesothelial cells showed telomerase reactivity. Thus, telomerase activity was specific for malignancy and it was always found only in malignant cells. The results suggest that telomerase activity measured with this in situ method can be a valuable complement in the assessment of malignancy in pleural effusions.     

Detection of telomerase expression in fine-needle aspirations and fluids

The cytologic examination of fine-needle aspirates and fluid specimens is plagued by a persistent false negative rate. The rate of false negative results will be decreased if sensitive molecular assays can be developed to detect cytologically malignant cells. The current study investigated telomerase expression as a potential marker of malignancy, using the telomeric repeat amplification protocol (TRAP) in fine-needle aspirates and fluid specimens. TRAP was performed on 24 fine-needle aspirate and 24 fluid specimens from different body sites and of different histological diagnoses. We found that 6 of 12 fine-needle aspirate specimens that were cytologically positive for malignant cells expressed telomerase activity, while no specimens that were cytologically suspicious for malignancy, atypical, or negative tested positive for telomerase activity. Of the fluid specimens, 4 of 6 cytologically positive cases and 1 of 18 cytologically negative cases expressed telomerase. Seven of eight telomerase negative, cytologically positive specimens contained only rare malignant cells in a very bloody background. Peripheral blood contamination is a possible pitfall in the TRAP assay, as applied in the current study, because the assay is standardized to protein concentration that may be derived from lysed red blood cells. We conclude that with further technical refinement, the TRAP assay could become a useful adjunct in the cytologic examination of fine-needle aspirates and fluid samples. Diagn. Cytopathol. 1998;18:431–436. © 1998 Wiley-Liss, Inc.     

Telomerase activity analyzed with TRAP in situ provides additional information in effusions remaining equivocal after immunocytochemistry and hyaluronan analysis

Cytology is central in the diagnosis of malignancy in effusions. Ancillary techniques, mainly immunocytochemistry, have considerably improved the sensitivity but some 10% of all cases remain equivocal and require the addition of new diagnostic modalities. We have previously shown that strong nuclear telomerase activity determined with Telomere Repeat Amplification Protocol (TRAP) in situ is specific for malignant cells and could be a candidate for an additional test. Thirty effusions remaining diagnostically equivocal after the use of immunocytochemistry and the determination of the hyaluronan content were reviewed and their TRAP in situ reactivity was related to the definitive diagnoses based on all available data. There were seven effusions from patients with definitive benign diagnoses and 23 effusions from patients with definitive malignant diagnoses. Strong telomerase activity was seen only in effusions from patients with definitive malignant diagnosis, all effusions from patients with benign disease lacking strong telomerase activity, whereas eight of the malignant cases, including three cases of epithelial mesothelioma, showed strong reactivity. Strong nuclear TRAP in situ reactivity was demonstrated only in effusions from patients with verified malignant disease. Although the study is small, it suggests that TRAP in situ activity provides diagnostic information in about one‐third of effusions remaining cytologically equivocal after the use of current ancillary techniques. The most striking diagnostic improvement appears to be gained in epithelial mesotheliomas. Diagn. Cytopathol. 2014;42:1051–1057. © 2014 Wiley Periodicals, Inc.     

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.     

胸腹水细胞端粒酶活性定性和定量检测

目的探讨端粒酶活性定量检测在诊断良恶性胸腹水中的应用价值。方法采用TRAP-银染定性方法和rrRAP-PicoGreen定量方法,对102例已确诊患者的胸腹水细胞进行端粒酶活性分析。结果恶性胸腹水细胞端粒酶活性明显高于良性胸腹水细胞,其定性检测诊断率明显高于细胞病理学。乳腺癌患者胸腹水细胞的端粒酶活性明显高于卵巢癌、肝癌患者胸腹水细胞的端粒酶活性;肺癌患者胸腹水细胞端粒酶活性明显高于肝癌。在良性胸腹水中,感染性胸腹水细胞端粒酶活性高于非感染性胸腹水。结论恶性胸腹水细胞端粒酶活性明显升高。端粒酶活性定量检测较定性检测更敏感、简便,对良恶性胸腹水的诊断和鉴别诊断有一定应用价值。     

Telomere repeat amplification protocol (TRAP) in situ reveals telomerase activity in three cell types in effusions: malignant cells, proliferative mesothelial cells, and lymphocytes.

Telomerase Repeat Amplification Protocol (TRAP) in situ was performed on cytospin preparations from 65 effusions from the serous cavities (45 pleural and 19 ascitic fluids and one pericardial fluid) submitted for routine diagnosis and the results were correlated to cytological morphology. Three types of cells with nuclear fluorescence were identified: malignant cells, hyperplastic mesothelial cell and lymphocytes. Of 38 cytologically malignant effusions, 12 showed strong reactivity in all malignant cells, three strong reactivity in part of the malignant population, whereas 12 showed moderate reactivity in the whole and five in part of the malignant population, respectively. In five malignant effusions weak reactivity was found in all (one case) and in scattered (four cases) malignant cells. Two effusions contained telomerase-negative malignant cells. Two pleural and two ascitic fluids contained proliferative mesothelial cells with weak or, in one case, moderate reactivity. Lymphocytes usually showed weak telomerase activity. Telomerase was expressed in almost all malignant tumours metastatic to serous cavities. Heterogeneity in tumour populations was demonstrated, which may have diagnostic implications, especially in cytology. Weak or moderate reactivity was found in lymphocytes and in some mesothelial proliferations and may explain the low specificity for malignancy sometimes obtained with the TRAP extract method. The weak reactivity found in lymphocytes may reduce the specificity when the extract method is used but causes no diagnostic problem with the TRAP in situ method.     

乳腺癌细针穿刺标本中端粒酶检测的诊断意义

目的 检测乳腺癌细针穿刺细胞标本中的端粒酶,探讨其在乳腺癌辅助诊断中的临床意义。方法 采用聚合酶链反应－端粒末端重复序列技术检测９９例乳腺细针穿刺标本（８３例乳腺癌、１２例乳腺良性病变和４例乳腺炎）中的端粒酶。     

Real-time quantitative analysis of telomerase activity in breast tumor specimens using a highly specific and sensitive fluorescent-based assay.

Telomerase activity has been associated with almost 90% of malignant human cancers from a variety of tissue sources, making it one of the most prominent molecular cancer markers known to date. As such, telomerase has become a very attractive diagnostic and therapeutic target. The advent of the telomeric repeat amplification protocol (TRAP) has allowed for the semiquantitative detection of telomerase from limiting sample amounts. Both the standard TRAP assay and a real-time assay using Amplifluor technology with primers designed specifically for telomerase activity amplification were used to quantitatively assess telomerase activity in primary tumors and tumor-derived cell lines. We have adapted the recently developed TRAPeze XL telomerase detection kit (Intergen, Gaithersburg, MD) for use with real-time polymerase chain reaction for more accurate quantification and high-throughput capabilities. In doing so, the reliability, assay time, and accuracy of quantitation have all been dramatically improved. A comparison of the quantitative analysis for the standard TRAP assay versus the real-time assay using 19 breast tumors revealed telomerase quantitation and standardization using the real-time assay was superior to the standard assay. Our data suggest that this assay will be useful for clinical and research studies involving detection of telomerase activity as it relates to cancer diagnosis.     

Comparison of the telomeric repeat amplification protocol (TRAP) to the new TRAP-eze telomerase detection kit

The ribonucleoprotein, telomerase, is believed to be responsible for the maintenance of telomere length in immortal and cancer cells. A PCR-based assay for the detection of telomerase activity (TRAP assay: telomeric repeat amplification protocol) was developed, allowing fast and efficient detection of telomerase activity when sample amounts are limiting. Of the thousands of primary human tumors examined using the TRAP assay, almost 90% have been shown to exhibit telomerase activity. Thus, for the early detection of cancer and for the rapid screening of compounds and drugs in cancer therapeutics, methods for the detection of telomerase activity are rapidly emerging. The recently developed TRAP-eze^TM kit from Oncor, Inc. gives increased sensitivity with decrease sample processing time, allowing improved detection of telomerase activity in a large number of samples. In the present study, we have addressed some of the technical aspects and limitations of critical importance for reproducibility, reliability, and linearity of the standard TRAP assay and the TRAP-eze^TM kit using cell culture and clinical materials.Abbreviations RP modified reverse primer (CX) for PCR amplification of telomerase products - AEBSF 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride - CHAPS 3-[(3-cholamidopropyl)dimethylammoniol-1-propanesulfonate - CX reverse primer for PCR amplification of telomerase products for standard TRAP - DEPC diethyl pyrocabonate - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid - HPLC high pressure liquid chromatography - ITAS internal telomerase assay standard - PCR polymerase chain reaction - T4g32 protein T4 gene 32 protein - TRAP telomeric repeat amplification protocol - TRF terminal restriction fragment - TS primer that serves as a substrate for telomerase     

Non-radioactive measurement of telomerase activity in human bladder cancer,bladder washings,and in urine

Early diagnosis is still the most important prerequisite for successful cancer treatment and this holds true for bladder cancer. Urine cytology is commonly used as a non-invasive screening procedure for the detection of bladder carcinoma, but this method is labour-intensive and often generates false-negative results. The ribonucleoprotein telomerase appears to be a promising new cancer marker, since its activity has been reported to correlate with indefinite growth. The aim of this study was to investigate whether telomerase activity can be detected in bladder cancer and in corresponding bladder washings. For this purpose, a sensitive non-radioactive TRAP (telomeric repeat amplification protocol) detection system was developed. With this technique, telomerase activity was found in 95 per cent of the carcinomas (n=20), in 70 per cent of the corresponding bladder washings, but in none of the urine samples obtained from patients with bladder carcinoma. No telomerase activity was detectable in normal urothelium or in samples from dysplastic urothelium. The data obtained from bladder washings show that superficial carcinoma cells released into the bladder still harbour telomerase activity. The absence of telomerase activity in voided urine is thus most likely due to degradation or inactivation under these conditions. The high rate of telomerase activity in bladder carcinoma indicates that the activation of telomerase is a common step in the tumourigenesis of bladder cancer. © 1998 John Wiley & Sons, Ltd.     

Chondrosarcoma is not characterized by detectable telomerase activity

Reactivation of telomerase, an enzyme which elongates human telomeres, is associated with cell immortilization. In approximately 90% of malignant tumours telomerase activity can be demonstrated, whereas in benign tumours it is mostly absent. Chondrosarcomas are relatively rare malignant cartilaginous neoplasms. A small number of chondrosarcomas located centrally in bone arise secondarily to an enchondroma, while the majority of chondrosarcomas developing from the surface arise within the cartilage cap of an osteochondroma. The histological distinction between a benign lesion and low-grade chondrosarcoma is generally considered difficult. To investigate whether the progression towards chondrosarcoma is characterized by reactivation of telomerase activity, this study determined telomerase activity in ten enchondromas, five osteochondromas, and 37 chondrosarcomas using the TRAP assay. In all tumour samples except one, telomerase activity was absent. By adding tumour lysates to the positive control, an increasing inhibition of telomerase activity was found with an increasing chondroid matrix, suggesting that it may contain inhibitory factors. Inhibition due to endogenous RNAse or Taq-polymerase inhibitors was excluded. The lack of detectable telomerase activity in the high-grade component of a dedifferentiated chondrosarcoma without matrix favours the possibility that telomerase is truly absent. Either its true absence or inhibitory effects disabling telomerase detection exclude the telomerase TRAP assay as a diagnostic tool in the differential diagnosis of benign and low-grade malignant cartilaginous tumours.     

Evaluation of telomerase in the development and progression of colon cancer

Telomerase activity, a cardinal requirement for immortalization, is a crucial step in the development of cancer and has been studied in many kinds of malignant tumours for clinical diagnostic and/or prognostic utilities. Using a PCR-based TRAP assay, we investigated telomerase activity in 8 adenomatous polyps, 9 dysplastic polyps, and in 36 paired cancer-normal mucosa specimens, one liver and one spleen metastasis from patients resected for sporadic colorectal cancer. Telomerase was absent or very low in normal mucosa and in adenomatous polyps. Dysplastic polyps and adenocarcinoma samples showed telomerase activity, with higher levels in cancer tissues compared to dysplastic lesions. A high telomerase activity was shown to be associated with late-staged cancers and metastasis, providing arguments supporting the role of telomerase not only in the development but also in the progression of colorectal carcinoma. Moreover, telomerase evaluation may help to confirm the malignant transformation in polypoid colorectal lesions with different levels of dysplastic alterations.     

Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.

AIMS/BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant tumours, but is inactive in normal somatic cells except for male germ cells and proliferating stem cells. Thus, the measurement of telomerase activity in tissue samples may provide useful diagnostic and prognostic information. The aim of this study was to determine whether telomerase expression is useful for the detection of occult malignant cells in lymph nodes. METHODS: Telomerase activity was compared with histological findings in 123 surgically removed lymph nodes submitted for routine or frozen section diagnosis. Telomerase activity was measured using a modified, semi-quantitative PCR-based telomeric repeat amplification protocol (TRAP). The assay was adapted for single 5 microns OCT embedded cryostat sections. In either fresh tissues or cryostat sections, normalised activity was linear when compared with protein concentration. Furthermore, using an in situ hybridisation method, the human telomerase RNA (hTR) component was measured in a subset of negative and positive nodes. RESULTS: Most (96%) of the 97 histologically negative nodes expressed low levels of activity (mean value of positive samples = 3.0 units/microgram protein) which may be derived from activated lymphocytes that express telomerase activity. All 26 malignant nodes (17 metastases, nine lymphomas) expressed telomerase (mean value = 17.8 units/microgram protein). The rank order levels between the two groups differed significantly (p = 0.0002). In situ results showed clearly that the hTR was expressed relatively highly in metastatic cancer cells and at lower levels in germinal centres of secondary follicles. CONCLUSIONS: Although expression of telomerase by activated lymphocytes may limit its usefulness, measurement of enzyme activity combined with detection of hTR using in situ hybridisation may assist in the histopathological diagnosis of lymph nodes.     

Telomerase activation in colorectal carcinogenesis.

Telomerase activity has been detected in germ cells as well as in the developing embryo. Activity is no longer detectable in most somatic cells of the neonate, although low levels of activity persist in regenerative tissues. Telomerase has been found to be reactivated or up-regulated in the majority of cancers. The colorectal adenoma-carcinoma sequence is one of the best-characterized models of multistep tumourigenesis and is thus suitable for determining at which stage telomerase is activated. Telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay in 96 cases of colorectal tissues, including 50 carcinomas, 31 adenomas, and 15 normal colonic tissues. For each case, histological diagnosis and telomerase activity were determined on consecutive frozen sections. In order to reduce the chance of a false-negative TRAP assay due to RNA degradation, the integrity of rRNA in the tissues was verified in each case. Twenty-five carcinomas, 30 adenomas, and all of the 15 normal colorectal mucosal samples showed no or only partial rRNA degradation and only in these cases was the TRAP assay interpreted. None of the normal tissues exhibited telomerase activity. In contrast, all of the 25 cancers and 47 per cent (14/30) of the adenomas were positive. In adenomas, telomerase activation was highly significantly related to the grade of dysplasia (p< 0.0001). All adenomas which contained high-grade dysplasia revealed telomerase activity, whereas telomerase activity was detectable in only 20 per cent (4/20) of cases with exclusively low-grade dysplasia. These results indicate that telomerase activation, which may be an obligatory step in colorectal carcinogenesis, occurs in the progression from low-grade to high-grade dysplasia in adenomas. Furthermore, in the adenoma-carcinoma sequence, telomerase activation seems to occur later than K- ras mutation but earlier than p53 mutation.     

Immunocytochemical studies of epithelial markers in pleural and peritoneal effusions as an adjunct to cytologic diagnosis

Monoclonal antibodies were used in an indirect immunoperoxidase assay to investigate 83 smears of pleural or peritoneal effusions for expression of the epithelial markers MAM-6, MAM-3, and carcinoembryonic antigen (CEA). All but one smears containing tumor cells according to the evaluation of H&E preparations were positive for one, two or all three markers, the exception being a malignant melanoma. Three of 5 cases, suspicious by routine cytology, exhibited marker expression in a different number of cells and thus confirmed the cytological diagnosis. Five of 63 cytologically negative smears exhibited single cells or small cell clusters positive for up to three markers. Four of these patients were found to suffer from metastatic cancer, as established by clinical follow-up and subsequent biopsy or autopsy, respectively. Felty's syndrome and concomitant serositis were diagnosed and confirmed by autopsy in one of the MAM-6 positive cases with negative cytology. The comparatively strong MAM-6 expression in some mesothelial cells of this patient might have been induced by abnormal stimulation due to the rheumatic disease. The results of this study encourage wider use of immunocytochemistry as an adjunct to cytological diagnosis in effusions.