Comparison of the RNA-amplification based methods RT–PCR and NASBA for the detection of circulating tumour cells

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Abstract
Increasingly, reverse transcriptase polymerase chain reaction (RT–PCR) is used to detect clinically significant tumour cells in blood or bone marrow. This may result in a redefinition of disease-free and clinical relapse. However, its clinical utility may be limited by lack of automation or reproducibility. Recent studies have suggested nucleic acid sequence-based amplification of target RNA may be more robust. In this study, nucleic acid sequence-based amplification was established to detect melanoma, colorectal and prostate cancer cells. Nucleic acid sequence-based amplification and RT–PCR both successfully amplified target RNA in peripheral blood samples from patients with melanoma and colorectal cancer, but only RT–PCR detected PSA in blood samples from patients with prostate cancer. There was relatively good agreement between sample replicates analyzed by RT–PCR (Kappa values of one for tyrosinase, 0.67 for CK-20 and one for PSA), but less agreement when analyzed by nucleic acid sequence-based amplification. This may limit the routine use of NASBA for the detection of clinically significant disease. In summary, RT–PCR appears at present to be the most reliable and reproducible method for the detection of low-level disease in cancer patients, although prospective studies are warranted to assess the clinical utility of different molecular diagnostic methods.