The Analysis of Tyrosinase-Specific mRNA in Blood Samples of Melanoma Patients by RT-PCR Is Not a Useful Test for Metastic Tumor Progression

Abstract

Reverse transcription (RT) of the tyrosinase mRNA and specific cDNA amplification by nested polymerase chain reactin (PCR) have been reported to facilitate the early detection of circulating tumor cells in melanoma patients. The significance and practical value of this procedure for the diagnosis of tumor dissemination in melanoma patients are unclear. In the current study we analyzed peripheral blood samples of 65 melanoma patients of different clinical stages for the presence of tyrosinase mRNA by RT-PCR using nested oligonucleotide primers specific for tyrosinase cDNA. Furthermore, blood samples were evaluated for tumor cell growth by cell culture assays in vitro. No tyrosinase mRNA was detectable in blood samples of 26 patients with primary melanoma and 16 patients with regional lymph node metastases. In five of 13 patients with visceral metastases we found at least one blood sample positive for tyrosinase mRNA during a 2-to 4-mo interval. Analyses of different blood samples of patients with visceral metastases taken in a 2-h interval, furthermore, indicate that tumor cells only transiently persist in the peripheral blood. We obtained in vitro proliferating melanoma cells from two blood samples derived from different patients with visceral melanoma metastases. This demonstrates that viable melanoma cells indeed circulate in the peripheral blood with retained proliferative capacity in vitro. The analysis of blood samples by RT-PCR for tyrosinase mRNA, however, is not suitable for the early detection of tumor progression in melanoma patients.

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 this study, relative quantity of tumour cells correlated with response to treatment. In addition, the same group found in this (Brossart et al, 1995) and in previous studies (Brossart et al, 1993(Brossart et al, , 1994 higher percentages of tyrosinase PCR-positive stage IV patients, incidentally reached by others (Mellado et al, 1996), but not in the majority of studies using tyrosinase PCR to detect circulating melanoma cells (Battayani et al, 1995;Foss et al, 1995;Hoon et al, 1995;Kunter et al, 1996;Pittman et al, 1996;Stevens et al, 1996;Ghossein et al, 1998;Glaser et al, 1997;Reinhold et al, 1997;Farthmann et al, 1998). Apart from technical differences among groups who use the tyrosinase PCR for the detection of circulating melanoma cells, differences in number of stage IV PCR-positive melanoma patients can be explained by differences in stage IV patients among the groups.…”

“…Second, in a recent EORTC quality control study, the reproducibility of the detection of tyrosinase transcripts in blood samples which were spiked with known amounts of melanoma tumour cells was poor, with three out of seven participating study groups scoring false positive results (Keilholz et al, 1998). Third, when taking various blood samples from one patient on one day, only some of them were found positive (Reinhold et al, 1997). Fourth, results from two blood samples taken at the same time were hardly reproducible (Farthmann et al, 1998).…”

Tyrosinase expression in the peripheral blood of stage III melanoma patients is associated with a poor prognosis: a clinical follow-up study of 110 patients

“…Our data do not provide more insights as to the origin of metastatic spreading in tyrosinase negative patients. Even if it cannot be excluded that the failure to detect circulating melanoma cells may depend on the sensitivity of the technique, the literature data show that tumour cells persist only transiently in the peripheral blood, as demonstrated by sequential analyses taken at a few hour-intervals from the same patients (Reinhold et al, 1997). The intermittent shedding of tyrosinase-positive cells in the bloodstream may, therefore, explain the discrepancies observed in patients with visceral relapses.…”

Limitations of the nested reverse transcriptase polymerase chain reaction on tyrosinase for the detection of malignant melanoma micrometastases in lymph nodes

“…Although clinical parameters such as lymph node involvement and thickness of the tumour are helpful, other diagnostic tools are urgently needed to allow for optimal prediction of the clinical course and choice of treatment. For this purpose efforts have been made to detect micrometastases in blood (Brossart et al, 1995;Foss et al, 1995;Hoon et al, 1995;Buzaid and Balch 1996;Mellado et al, 1996;Gläser et al, 1997;Jung et al, 1997;Reinhold et al, 1997;Farthmann et al, 1998;Ghossein et al, 1998;Curry et al, 1998;De Vries et al, 1999;Palmieri et al, 1999) and in the regional lymph nodes by immunohistochemistry or reverse transcriptase polymerase chain reaction (RT-PCR) (Battyani et al, 1993;Wang et al, 1994;Rankin, 1996;Van der Velde-Zimmermann et al, 1996;Blaheta et al, 1998;Goydos et al, 1998;Hatta et al, 1998;Bieligk et al, 1999).…”

“…The initial report led to a number of more detailed investigations regarding the presence of tyrosinase RNA in peripheral blood of melanoma patients (Brossart et al, 1995;Foss et al, 1995;Hoon et al, 1995;Buzaid and Balch, 1996;Mellado et al, 1996;Gläser et al, 1997;Jung et al, 1997;Reinhold et al, 1997;Curry et al, 1998;Farthmann et al, 1998;Ghossein et al 1998;Palmieri et al, 1999;De Vries et al, 1999). The published tyrosinase mRNA RT-PCR sensitivities in peripheral blood among stage III melanoma patients range from 0% to 100%.…”