Detection of melanoma cells in peripheral blood by tyrosinase RT-PCR in patients with malignant melanoma

Abstract

DETECTION OF MELANOMA CELLS IN PERIPHERAL BLOOD BY TYROSINASE RT-PCR IN PATIENTS WITH MALIGNANT MELANOMA &Qt Hochbem. Michal Lotem. Eitan Shiloni and Claes D. Enk. Departments of Dannatolcgy and Oncology, Hadassah Medical Center, Jerusalem, and Department of Surgery, The Carmel Hospital, Hafa, Israel. Identiioation of tyrrxinase expressing cells in the peripheral blood of patients with malignant melanoma is an early indicator of met&&c spreading, since melanocytes do not ordinarily circulata. To establish the parameters of the RT-PCR assay, sarial dilutions of tyrosinasepositive SK28 melanoma cells in normal peripheral blood were exploited: When using nested primers, tymsinase gene transcfiption was often d&&ad in the negative controls, probably due to illegitimate transcription or contamination. When using non-nested pimers for amplification followed by Southam blots for verification, wa could reproducibly identii down to 100 SK28 calls in 5 ml of peripheral blood with no detectable message in tha negative controls. To establish tha validity of the assay in a clinical setting, w tested the blood of 17 patients with malignant melanoma and 10 patients with non-malancvna skin diseases or healthy amtrols: All 15 patients with stage Ill / IV disease tested positive, whereas only 1 of the 2 patients with stage I I II disease was positive. Three of the controls tastad positive as well. Thus the sensitivity was 100% for advanced diseasa, whereas the speciftcity was only 70 %. We conclude that a negative test result is a reliable measure for the absence of m&static disease, and suggast that the spwificity of ths assay might be improved by use of additional tumor markars.