Nested Quantitative Real Time PCR for Detection of Occult Tumor Cells

Abstract

Quantification of tumor mRNA markers expressed by occult circulating tumor cells may be of prognostic value in a variety of neoplasms and disease stages. We therefore developed a novel real time nested polymerase chain reaction (PCR) assay to quantify rare transcripts using the light cycler system. Tyrosinase mRNA expressed by melanocytes and melanoma cells was used as a model. Ten-milliliter samples of ethylenediaminetetra-acetate (EDTA) blood from healthy volunteers were spiked with 10–10⁴ RVH melanoma cells. Following RNA extraction and cDNA synthesis, a nested PCR with specific primers was performed. Resonance energy transfer between fluorescently labeled hybridization probes specific for the amplified portion of the tyrosinase transcript allowed continuous monitoring of the second round of PCR. The number of preamplification cycles in the first round was varied between 20 and 35. Our results show that nested PCR revealed quantitative data, regardless of the number of cycles used in the first round. The range of real time data can span four logs of target mRNA concentrations (1–10³ tumor cells/ml of blood) and the x-intercepts of the log phases were always consistent with the amount of transcripts in the sample. These experiments indicate that nested PCR, which is much more sensitive than single-round PCR, is a useful tool for the quantification of rare transcripts — a result with important implications for the study of minimal residual disease. The assay is currently being adapted for other tumor types including leukemias as well as other solid tumors.

Supported by the Deutsche Krebshilfe, grant no. 70-2459-Ke2, the Sonnenfeldstiftung Berlin, and the EORTC Melanoma Cooperative Group.