Increased sensitivity for the detection of malignant melanoma cells in peripheral blood using an improved protocol for reverse transcription-polymerase chain reaction

Conflicting results have been obtained by various research groups using tyr osinase reverse transcription-polymerase chain reaction (RT-PCR) for detect ing micrometastases in the blood of melanoma patients, with positive result s ranging from 0 to 100% in disseminated melanoma, Methodological differenc es in the processing of blood samples may in part account for these discrep ancies. The aim of this study was to standardize and optimize the experimen tal conditions for RT-PCR detection of melanoma cells in peripheral blood. We analysed the effect of different parameters of sample processing on the sensitivity of the tyrosinase RT-PCR using peripheral blood spiked with def ined numbers of SKMEL28 melanoma cells. Purification of the mononuclear cel l fraction using a Ficoll gradient with a density of 1.077 g/mL prior to RN A isolation gave the highest sensitivity, with the detection of two SKMEL28 cells in 5 mL, of blood. In addition, the RNA isolation method and the kin d of RT enzyme used had a significant impact on the sensitivity and reprodu cibility of tyrosinase detection, whereas variations in the PCR conditions had only a minor influence. Furthermore, we showed that amplification of Me lanA in addition to tyrosinase resulted in an approximately 10% enhanced se nsitivity of melanoma cell detection, whereas gp1w00/pMel17 and MUC18 gene products were also detected in blood from non-melanoma patients, MelanA cou ld serve as a sensitive marker in addition to tyrosinase for detecting micr ometastases.