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Microphtalmia transcription factor and tyrosinase as markers for detection of melanoma cells in the blood of melanoma patients by RT-PCR

Reverse trasnscription – polymerase chain reaction (RT-PCR) for tyrosinase mRNA is specific and sensitive method for detection of circulating melanoma cells in peripheral blood of melanoma patients. Still, the real clinical value of tyrosinase as a marker for that purpose is limited. Therefore, other markers for RT-PCR detection of circulating melanoma cells are being investigated. Microphthalmia transcription factor (MITF) is one such molecule that might serve as a marker for RT-PCR detection of circulating melanoma cells, although it has been used and investigated so far only as an immunohistochemical melanoma marker. The aim of this study was to investigate and compare sensitivity and specificity of microphthalmia transcription factor (MITF) and tyrosinase as markers for RT-PCR detection of circulating melanoma cells on peripheral blood samples of metastatic melanoma patients. Blood samples from 33 melanoma patients with metastatic melanoma were analyzed. RNA was isolated from mononuclear cell fraction of the blood and it was reverse transcribed into the cDNA. The cDNA was then assayed by PCR for the expression of tyrosinase and MITF. Healthy volunteers were used as negative controls. We were able to detect single HBL melanoma cell on 0.82x106 peripheral blood leukocytes when using tyrosinase, as well as when using MITF. Among 20 blood samples from patients with regional lymph node metastases, 2 were positive for both tyrosinase and MITF, 5 for tyrosinase only, and 3 for MITF only. Among 13 blood samples from patients with distant metastases, 3 were positive for both tyrosinase and MITF, 2 for tyrosinase only, and 2 for MITF only. The use of MITF in addition to tyrosinase resulted in a 15% enhanced sensitivity of melanoma cells detection in comparison with tyrosinase only. All samples from healthy volunteers were negative for both tyrosinase and MITF. In our study we showed high sensitivity and specificity of RT-PCR for both tyrosinase and MITF in detection of circulating melanoma cells in peripheral blood of melanoma patients. Our results suggest that using MITF in addition to tyrosinase improves the detection of circulating melanoma cells.

MITF; tyrosinase; RT-PCR; melanoma

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