Tyrosinase mRNA levels in the blood of uveal melanoma patients: correlation with the number of circulating tumor cells and tumor progression Pamela Pinzani a , Cinzia Mazzini c , Francesca Salvianti a , Daniela Massi b , Raffaella Grifoni d , Costanza Paoletti d , Francesca Ucci c , Elena Molinara d , Claudio Orlando a , Mario Pazzagli a and Bruno Neri d We measured tyrosinase mRNA levels by real-time quantitative reverse transcription-PCR (qRT-PCR), in the blood of patients with uveal melanoma. Results were correlated with clinical data and, in a subgroup of patients, with the number of circulating tumor cells (CTC) assessed using isolation by size of epithelial tumor cells (ISET). Forty-one patients with uveal melanoma were longitudinally investigated over a period of 5 years. The standard curve of the qRT-PCR method used melanoma cell line SK-MEL-28, added to the blood of normal donors and it was calibrated on a synthetic RNA standard (1 SK- MEL-28 cell corresponding to 18 tyrosinase mRNA copies) to improve the procedural standardization to facilitate the comparison of data collected at different laboratories. Increased tyrosinase mRNA levels were found in at least one of the blood samples in 20 of 41 (49%) uveal melanoma patients (median 0.8 SK-MEL-28 cell equivalents/ml blood; range 0.1–14.4). A significant correlation was found between mRNA tyrosinase levels and tumor dimension (P < 0.01), disease-free and overall survival (P < 0.05). CTC were isolated by ISET in five of 16 patients (5.8, 2.33, 2.00, 1.25, and 0.75 CTC/ml of blood) and the corresponding tyrosinase mRNA levels were 2.13, 1.37, 0.83, 0.58, and 0.35 SK-MEL-28 cell equivalents/ml of blood. Tyrosinase was undetectable in 11 ISET-negative patients. Tyrosinase assay by qRT-PCR is a noninvasive method for the detection of tumor progression in uveal melanoma patients. The mRNA tyrosinase levels can be taken as an indirect parameter correlated to the number of CTC isolated from blood by ISET. Melanoma Res 20:303–310  c 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins. Melanoma Research 2010, 20:303–310 Keywords: circulating tumor cells, real-time RT-PCR, tyrosinase expression, uveal melanoma Departments of a Clinical Physiopathology, b Human Pathology and Oncology, c Department of Oto-neuro-ophthalmology, Eye Clinic and d Department of Oncology, Centre of Experimental and Clinical Oncology, University of Florence, Florence, Italy Correspondence to Pamela Pinzani, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50145 Florence, Italy Tel: + 39 554271441; fax: + 39 554271371; e-mail: p.pinzani@dfc.unifi.it Received 8 June 2009 Accepted 11 February 2010 Introduction The incidence of uveal melanoma is approximately six to seven cases/1 000 000 inhabitants [1,2] in the Caucasian population [3]. It is a slowly growing tumor, with a low mitotic index and apoptotic rate [4], often associated with an unfavorable outcome, with more than 50% of affected patients dying of the disease within 5–10 years [5]. Several clinical, histological, and genetic factors have been identified as important risk factors for local recurrence, metastasis development, and survival [6]. As most patients with uveal melanoma do not undergo surgery and are treated with radiotherapy, histological and genetic predictive factors are not usually investigated. Tumor cells of ocular melanoma disseminate through the blood stream, because of the absence of lymphatic vessels in the uveal tract. As blood-borne tumor cell dissemina- tion is the main mechanism responsible for distant meta- stases, with a marked tendency to metastasize to the liver, the hematological approach may be highly desirable for detecting systemic tumor cell spread [7]. In particular, molecular markers can be useful tools for the early detection of disseminated tumour cells in cancer patients [7,8], whereas PCR-based techniques have already been applied to the diagnosis of subclinical cancer diseases, spreading and monitoring of the treatment response [9]. Several molecular markers for the detection of dissemi- nated tumor cells in the blood of melanoma patients have been investigated [10–14] and, among them, tyrosinase (a key enzyme involved in the biosynthesis of melanin expressed by melanocytes and melanoma cells) appears to be one of the most effective, as its transcripts are generally not detected in blood samples from healthy donors [15]. Tyrosinase has been investigated mainly on cutaneous melanoma, but some studies [16–18] have also evaluated tyrosinase mRNA levels in patients with uveal melanoma. More recent reports [19] indicate that high values of circulating tyrosinase mRNA are frequently found in patients with melanoma liver metastases. Moreover, tyrosinase is an independent prognostic factor in patients with primary uveal melanoma at high risk for Original article 303 0960-8931  c 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/CMR.0b013e32833906e3 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.