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Clinical Chemistry 53: 53-61, 2007. First published November 27, 2006; 10.1373/clinchem.2006.073015
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(Clinical Chemistry. 2007;53:53-61.)
© 2007 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Real-Time RT-PCR Quantification of Human Telomerase Reverse Transcriptase Splice Variants in Tumor Cell Lines and Non–Small Cell Lung Cancer

Eleni Mavrogiannou1, Areti Strati1, Aliki Stathopoulou1, Emily G. Tsaroucha2, Loukas Kaklamanis3 and Evi S. Lianidou1,a

1 Laboratory of Analytical Chemistry, University of Athens, Athens, Greece.
2 "Sotiria" General Hospital for Chest Diseases, Athens, Greece.
3 Department of Pathology, Onassis Cardiac Surgery Center, Athens, Greece.

aAddress correspondence to this author at: Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, 15771 Athens, Greece. Fax 30-210-7274750; e-mail lianidou{at}chem.uoa.gr.

Background: We developed and validated a real-time reverse transcription (RT)–PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants ({alpha}+ß+, {alpha}–ß+, {alpha}+ß–, {alpha}–ß–) in tumor cell lines and non–small cell lung cancer (NSCLC).

Methods: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript–specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients.

Results: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The {alpha}+ß– splice variant showed the highest expression and {alpha}–ß+ and {alpha}–ß– the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the {alpha}+ß– variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by {alpha}+ß+, with the {alpha}–ß+ and {alpha}–ß– splice variants having the lowest expression. In the NSCLC tumors, the {alpha}+ß+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival.

Conclusions: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT {alpha}+ß+ splice variant may be an independent negative prognostic factor for NSCLC patients.






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