Real-time telomerase assay of less-invasively collected esophageal cell samples
Introduction
The development of biomarkers, their validation and translation to clinical application is becoming increasingly important for the diagnostic community [1]. The search for cancer-associated molecules has led to the discovery of many molecular markers associated with clinical outcome [2], [3]. Among more than 100 validated cancer biomarkers, telomerase is unique [4]. Telomerase activity is the single most common cancer biomarker, detected in 80–90% of cancers [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. In some tissues, telomerase activity was detected at the preneoplastic or in situ stage, suggesting that telomerase activity measurements have potential for early detection applications and telomerase is increasingly being considered as a target for cancer prevention and therapeutics [16]. In other cases, telomerase activity increased with cancer progression, suggesting that telomerase activity measurements could function as a prognostic indicator of patient outcome.
Esophageal cancer is the sixth leading cause of cancer death worldwide [17]. Most cases occur in developing countries, are squamous cell carcinomas and are diagnosed at an advanced stage when therapy with curative intent is not possible. There is great interest in the development of a primary screening test to identify patients with premalignant or early malignant disease, which could be more readily cured. Studies from our group have demonstrated that squamous dysplasia is the precursor lesion for esophageal squamous cell carcinoma (ESCC) [18] and that these lesions can be observed during endoscopy with Lugol's iodine staining [19], but endoscopy is too invasive and too expensive to serve as a primary screening exam even in very high risk populations. Previous efforts to develop a less invasive and more cost-effective primary screening test for ESCC have focused on EBC, a process analogous to a pap smear, but to date this technique has had insufficient sensitivity for detecting squamous dysplasia and cancer [20]. It is evident that molecular high-throughput approaches are needed to develop a more sensitive, less invasive and objective test for mass screening of high-risk populations. Telomerase activity and hTERT mRNA appear to be absent in the outer epithelial layers of normal esophageal squamous epithelium, but they increase in preneoplastic lesions and squamous cell carcinomas [21], [22], so they may be useful markers in a molecular screening test for these lesions and they may improve the clinical utility of EBC exams. Advances in molecular biology have provided a greater understanding of cancer progression including the use of biomarkers of early detection and risk assessment [23]. Automated and high-throughput assays and reference materials have recently advanced the utility of telomerase as a diagnostic for such early detection [24], [25].
Early detection of disease is a major focus by which to improve outcomes for patients with solid tumors [3], [26], [27]. Further, biomarker discovery and validation is in great demand by the pharmaceutical and healthcare industries. High-throughput [28] and global scanning [29] methods that are low-cost and reliable clinical platforms are essential to analyze large cohort studies, which can statistically validate potential DNA, RNA and protein biomarkers for early detection assays. Standardized controls or reference materials will be essential for the acceptance of data collected from these new technology platforms by regulatory agencies.
In partnership with the National Cancer Institute's Early Detection Research Network (EDRN), the National Institute of Standards and Technology (NIST) evaluates analytical methods for the validation of biomarkers associated with the detection of cancer and optimizes high-throughput technologies for clinical applications [28], [29], [30], [31]. The mission is to validate biomarkers for reliable results and demonstrate proof-of-principle of their translation into clinical applications. Then, private sector or other government agencies can promote the use of the biomarkers and clinically relevant technologies in clinical trials and can incorporate NIST standards and guidelines to establish a diagnostic.
The combination of less-invasive collection techniques such as EBC and rapid diagnostic assays are central components for population screening [32]. It is critical that populations at-risk for cancer have a real-time based assay. Although automated TRAP assays have been developed, many difficulties with TRAP assays have prevented their wide acceptance in clinical settings. Several drawbacks include: TRAP assays are multi-step, time-consuming, and require extensive post-PCR processing to determine if the potential cancer sample has signal greater than the negative control and activity comparable to the positive control. These problems have been resolved with real-time TRAP assays.
Recently, we reported the development of a fluorescent-based, real-time assay to measure telomerase activity [24]. This one-step procedure simplifies the TRAP assay. RTTRAP is less cumbersome, maintains high-throughput, and obviates the need for extensive post-PCR processing. Further, RTTRAP compressed the assay time to 1 h as compared to at least 10 h with traditional protocols [24], [33]. In the current study we measured the telomerase activity of eight EBC specimens from a population at high risk for esophageal cancer and we compared these activity measurements to the number of hTERT mRNA transcripts and to other TRAP methods. Our findings suggest potential for RTTRAP techniques in telomerase-based primary screening assays using similar samples.
Section snippets
Materials and methods*
*Certain commercial equipment, instruments, materials, or companies are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation nor endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are the best available for the purpose.
Results
In this study, we evaluated the telomerase activity of cell extracts recovered from balloon cytology as a potential less-invasive diagnostic tool. The telomerase activity of each cell extract was measured by RTTRAP and also measured across several different TRAP technologies and platforms. TRAP methods, RApidTRAP and hTERT mRNA measurements, independently confirmed the results obtained from RTTRAP. Table 1 shows the characterization of the clinical samples using Real-Time detection, capillary
Discussion
In this study, we evaluated telomerase activity and hTERT mRNA in samples obtained by EBC in a population at high risk for ESCC. This was an initial step in evaluating the feasibility of screening for esophageal precursor lesions and early ESCC based on telomerase activity. The telomerase activity of each sample was measured in parallel by a new real-time TRAP assay (RTTRAP) and by other telomerase technologies and platforms. The RApidTRAP and hTERT mRNA measurements were concordant with the
Acknowledgements
This study was a collaborative effort between the National Institute of Standards and Technology (NIST), the National Cancer Institute (NCI), the NCI Early Detection Research Network (EDRN), and the Cancer Institute of the Chinese Academy of Medical Sciences. The study was supported in part by NCI Contract No. N01-RC-91019 and in part by the Intramural Research Programs of the NCI Division of Cancer Epidemiology and Genetics and the Center for Cancer Research. Clinical samples were received by
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Modulation of telomerase activity, bTERT and c-Myc induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin during Bovine Herpesvirus 1 infection in MDBK cells
2014, Toxicology in VitroCitation Excerpt :By comparing telomerase activity and bTERT protein level measurements, we observed a good correlation, showing a similar trend. These data were in agreement with previous studies carried out on MDBK cells (Fiorito et al., 2011) and on esophageal epithelial cells, describing an evident correlation between telomerase activity and the amount of cellular hTERT mRNA (McGruder et al., 2006). To determine if the regulation of c-Myc was also involved in telomerase down-regulation by TCDD, we carried out Western blot analysis for c-Myc, and we showed that dioxin significantly down-regulated c-Myc protein levels.
Label-free highly sensitive detection of telomerase activity in cancer cell by chemiluminescence imaging
2012, Molecular and Cellular Probes2,3,7,8-Tetrachlorodibenzo-p-dioxin induced autophagy in a bovine kidney cell line
2011, ToxicologyCitation Excerpt :In addition, by comparing telomerase activity and bTERT protein level measurements, we observed a good correlation, showing a similar trend. These data were in agreement with a previous study carried out on esophageal epithelial cells, describing an evident correlation between telomerase activity and the amount of cellular hTERT mRNA (McGruder et al., 2006). To determine if the regulation of c-Myc was also involved in telomerase down-regulation by TCDD, MDBK cells were treated with TCDD and total cell lysates were subjected to Western blot analysis.