RT-PCR for Tyrosinase-mRNA-Positive Cells in Peripheral Blood: Evaluation Strategy and Correlation with Known Prognostic Markers in 123 Melanoma Patients

Abstract

Reverse transcriptase polymerase chain reaction for the detection of tyrosinase-mRNA-positive cells in peripheral blood of melanoma patients, as a possible marker of hematogenous dissemination, has demonstrated varying detection rates. This study examined the sensitivity and reproducibility of the technique using a protocol of multiple polymerase chain reaction to determine circulating melanocytic cells. For each of the 123 melanoma patients included in this study, four nested polymerase chain reactions were performed from two blood specimens requiring both polymerase chain reactions from at least one blood sample to be positive to consider a patient as positive. Thus, a definitive result was obtained in 98% of the cases, whereas only 1.6% lacked conclusive findings. Thus, we found a correlation between the tyrosinase detection rate and the clinical stage. Circulating tyrosinase-mRNA-positive cells were detected in 13% of patients with primary tumor, 17% with regional skin/lymph node metastasis, and 44% with distant metastasis. Positivity also correlated with known melanoma progression markers such as gender, tumor thickness, and histologic type. Positive results were obtained more frequently in (i) men compared with women, (ii) patients with thick primary melanomas (> 4 mm: 38%) compared with those with thinner tumors (1.1-4 mm, 22%; < or = 1 mm, 5%), and (iii) patients with nonclassifiable (38%), nodular (34%), and occult primary melanomas (30%) compared with those with acrolentiginous (17%), superficial spreading (9%), or lentigo maligna melanoma (0%). These findings suggest that detection of tyrosinase-mRNA-positive cells in peripheral blood is not an adequate marker for identifying melanoma patients with distant metastasis. Reverse transcriptase polymerase chain positivity in early melanoma stages, however, as corresponding to other prognostic parameters, may indicate increased risk for the development of hematogenous metastasis and may be of value as a progression marker.

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

“…There is no agreement in the literature as to the percentage of positive tyrosinase determinations in stage III patients, with a wide range of values up to 60%, according to the different studies (Brossart et al, 1993;Kunter et al, 1996;Pittman et al, 1996;Glaser et al, 1997;Farthman et al, 1998;Proebstle et al, 2000;Reinhold et al, 2001). Our results showed a positive tyrosinase expression in 49% of patients.…”

“…Several studies (Curry et al, 1998;Farthman et al, 1998;Ghossein et al, 1998;Alao et al, 1999;Reinhold et al, 2001) have suggested a potential clinical use of the RT -PCR analysis of tyrosinase mRNA to detect circulating neoplastic cells in the peripheral blood of melanoma patients. In a previous paper , we demonstrated that the majority of untreated stage IV melanoma patients were positive, whereas chemotherapy administration was able to reduce tyrosinase rate, suggesting that tyrosinase may provide a measure of therapy response and be a predictor of disease evolution.…”

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).…”

“…Among these techniques the nested RT-PCR is the most sensitive method: in spike experiments the detection limit ranges from 1 to 10 tumour cells in 10 7 peripheral blood lymphocytes (PBLs) (Hoon et al, 1995;Gläser et al, 1997;Jung et al, 1997;Farthmann et al, 1998;Palmieri et al, 1999), whereas for immunohistochemistry the detection limit is 1 tumour cell in 10 5 normal cells (Hatta et al, 1998).…”

“…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%.…”