title: Optimization of the RT-PCR technique to detect melanoma cells in peripheral blood.
abstract: Detection of melanoma cells in the peripheral blood of melanoma patients by reverse transcription polymerase chain reaction techniques has demonstrated varying detection rates. This study examined the sensitivity of the technique by employing a modification of the currently used protocols to detect mRNA markers. RT-PCR of tyrosinase and MART-1 was performed after poly A+-RNA isolation from unmanipulated whole blood lysed in the presence of nuclease inhibitors. We found a preclinical sensitivity of 1 GR-M melanoma cell spiked in 1 ml of blood. The clinical sensitivity was tested by studying 22 melanoma patients with advanced disease. The rate of positivity in all patients was 63.6% for tyrosinase, 50% for MRT-1 and 77.3% for at least one molecular marker. This figure increased to 89.5% when considering only those patients with evident macroscopic disease. We can conclude that the technical modifications introduced in this protocol significantly increased the clinical sensitivity compared with other published methods.
authors.affiliation: Department of Functional Biology, Hospital Central de Asturias, Universidad de Oviedo, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Suárez
authors.forename: Joaquín
authors.initials: J
authors.lastname: Fra
authors.forename: Rebeca
authors.initials: R
authors.lastname: Alonso
authors.forename: Rosa
authors.initials: R
authors.lastname: Sánchez
authors.forename: Angel J
authors.initials: AJ
authors.lastname: Lacave
authors.forename: Carmen
authors.initials: C
authors.lastname: Gutiérrez
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000951:Antigens, Neoplasm; D005260:Female; D006801:Humans; D058965:MART-1 Antigen; D008297:Male; D008545:Melanoma / Q000097:blood* / Q000473:pathology*; D008875:Middle Aged; D014442:Monophenol Monooxygenase / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D000951:Antigens, Neoplasm; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D014442:Monophenol Monooxygenase