Reproducibility of detection of tyrosinase and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR

In recent years, large discrepancies were described in the success rate of the tyrosinase reverse transcription polymerase chain reaction (RT-PCR) for detecting melanoma cells in the peripheral blood of melanoma patients. We present a quality control study in which we analysed the reproducibility of detection of tyrosinase and MART-I transcripts in 106 blood samples from 6 8 melanoma patients (mainly stages III and IV). With this study, we aimed t o improve insight in the reproducibility of a RT-PCR for the detection of ( minimal) amounts of circulating melanoma cells. We performed two reverse tr anscriptions on each mRNA sample and performed tyrosinase and MART-1 nested PCRs in duplicate per cDNA sample. Thus, four tyrosinase and four MART-I m easurements were performed per blood sample. In our study, the majority of blood samples was negative for tyrosinase (80%) or MART-I (66%). Only four samples were positive in all four determinations for tyrosinase and seven f or MART-1. Variable results (1-3 times positive results) were obtained for tyrosinase and MART-1 in 16% and 27% respectively MART-1 PCR had a better p erformance than tyrosinase PCR, Sensitivity increased when both markers wer e used. We reasoned that the low number of melanoma marker PCR-positive blo od samples can be explained by differences in mRNA quality. By using real-t ime quantitative PCR, we found that this was not the case: amplification of porphobilinogen deaminase (PBGD), a low copy household gene, was not diffe rent in blood samples in which a melanoma marker was not detected from grou ps in which this marker was detected more or less consistently (1-4 times). When applying real-time quantitative PCR far tyrosinase and MART-I, we fou nd that a low amount of SK-MEL-28 cell equivalents was present in the blood of melanoma patients, with a higher number of equivalents in the group wit h a consistently positive result. We conclude that low reproducibility of a repeated assay for the detection of circulating melanoma cells is not caus ed by differences in mRNA quality between the samples, but due to low numbe rs of amplifiable target mRNA molecules in the mRNA sample. Use of more tha n one marker and repetition of the assay will increase the probability of f inding positive PCR results.