Analytical Validation of Telomerase Activity for Cancer Early Detection: TRAP/PCR-CE and hTERT mRNA Quantification Assay for High-Throughput Screening of Tumor Cells

Abstract

Activation of telomerase plays a critical role in unlimited proliferation and immortalization of cells. Telomerase activity has been shown to correlate with tumor progression, indicating that tumors expressing this enzyme possess aggressive clinical behavior and that telomerase activity may be a useful biomarker for early detection of cancer. However, measurements of telomerase activity by current methods such as telomeric repeat amplification protocol (TRAP)/polymerase chain reaction (PCR) or antibody-based radioimmunoassay (RIA) are low-throughput and not robust enough to easily accommodate the required statistical analysis to determine whether telomerase activity is a practical biomarker. As part of the National Cancer Institute Early Detection Research Network of analytical validation, we have developed a robot assisted TRAP assay (RApidTRAP) of telomerase, a potential biomarker for cancer early detection. Measurements of human telomerase reverse transcriptase catalytic subunit (hTERT) mRNA were performed in concert with measurement of telomerase activity. For this purpose we determined hTERT mRNA concentration and telomerase activity in human normal (RPE-28) and cancer (A549) cell lines as well as in human serum (SRM 1951A). Telomerase activity measurements were made using the TRAP/PCR capillary electrophoresis (CE) method on (50 to 1000) cells/reaction isolated from cell extracts. Measurement of hTERT mRNA was made using specific primers and probes on a LightCycler in the range of (10 to 7000) cells/reaction. Comparison of high-throughput telomerase activity measurements using the robot and those performed manually were consistent in sensitivity and reproducibility. Using this combination of telomerase activity and hTERT mRNA measurements, the automated system improved efficiency over traditional TRAP/PCR methods.