Detection of MetastaticTumor Cells in Blood by Reverse Transcriptase-Polymerase Chain Reaction

In oncology, the correct assessment of tumor stage is crucial information, as it relates to disease-free interval and prognosis. Routine staging modalities use histopathology to evaluate the presence or absence of metastasis mainly in tissues such as lymph nodes, as described in Chapter 1 by Roskell and Buley. Retrospective analysis using fine sectioning techniques has shown that approx 30% of tumor patients may be understaged at the time of diagnosis (1 ) Since the early 1980s, immunohistochemical and -cytochemical methods have been devised to increase the sensitivity in detecting small tumor loads or single tumor cells (2 ) Clinical studies using monoclonal antibodies against cytokeratins, intermediate filament proteins characterizing epithelial cells and thus carcinomas, have shown that the presence of such cells in the bone marrow is an independent prognostic marker of disease-free and overall survival (3 -5 ). The methodology for this approach is given in Chapter 5 by Braun and Pantel. With the advent of extremely powerful nucleic acid amplification and detection methods a significant increase in analytical and diagnostic sensitivity of tumor cell detection has been achieved. Such micrometastasis assays detect messenger RNA (mRNA) molecules transcribed from genes active in the cancer cell, but not the non-malignant cellular components otherwise present in the sample material. In this technique, a first strand cDNA copy is first produced from the RNA molecules using a reverse transcriptase (RT). The RT step is then followed by an enzymatic exponential amplification reaction, most commonly performed using the polymerase chain reaction (PCR).