Detection of Spiked Uveal Melanoma Cells in Peripheral Blood of Healthy Volunteers

Abstract

Abstract: : Purpose: The identification of circulating malignant cells (CMCs) in uveal melanoma (UM) patients is the most important indicator of patients at risk of developing metastatic disease. Detection of CMCs using RT-PCR has been investigated in several malignancies to identify these high-risk patients who may benefit from adjuvant therapy. Tyrosinase and Melan A have been successfully used as RT-PCR markers to detect CMCs in skin melanomas. To test the sensitivity of RT-PCR analysis for the detection of CMCs in UM, blood of normal volunteers was spiked with human UM cells Methods: Twelve peripheral blood samples were divided in 4 groups of 3 samples each. Each group was spiked with 1 of 4 human uveal melanoma cell lines (SP6.5, MKTBR, UW-1, and OCM-1). Each sample (14ml of blood) was divided in 7 aliquots of 2 ml each. Six aliquots were spiked with increasing concentrations of UM cells (10, 10²,10³,10⁴,10⁵, and 10⁶). One aliquot was used as negative control. Blood was processed immediately for purification of the mononuclear layer (lymphocytes and melanoma cells) by adding Ficoll either 1.077g/ml or 400 gradient. RT-PCR was performed using both Tyrosinase and Melan A as markers. Total lymphocyte count in the original blood samples was compared with the final count in the mononuclear layer, measured by Trypan Blue, to determine the efficacy of the extraction (reported efficacy of extraction: 50 ± 10%). Results: All samples were positive for both markers at 10⁵ and 10⁶ cells. Eight samples were positive when spiked with 10⁴ cells, and 3 were positive at 10³ cells. Both markers were equally sensitive at detecting cells and no difference in detection was found between the four cell lines used. Results were obtained using 1.077g/mL Ficoll as no cells were detected using 400 Ficoll. The efficacy of the extraction was 65% (range: 52-77 %). Conclusions: RT-PCR, using either tyrosinase or Melan A as markers, is a sensitive and reliable method for the detection of UM cells. Identification of CMCs in peripheral blood of UM patients by RT-PCR may be a useful new method to monitor high-risk patients. Peripheral blood is a readily available source of information that may be easily analyzed in a clinical setting. The detection of subclinical spread of disease may enable adjuvant therapies to be initiated at early stages of dissemination.

Keywords: clinical (human) or epidemiologic studies: ris • clinical (human) or epidemiologic studies: sys • melanoma