Purpose: The finding of melanoma cells in the peripheral blood, thus far mainly inferred from the PCR-based demonstration of tyrosinase mRNA, has been associated with metastatic melanoma. Neither the malignant nature nor the prognostic significance of circulating cells could be established. To address this question, we analyzed immunomagnetically isolated circulating melanoma cells for chromosomal aberrations and performed a clinical follow-up study of the enrolled patients.Experimental Design: In a prospective study, blood samples were taken from 164 melanoma patients and 50 donors without malignant disease. Circulating melanoma cells were enriched by immunomagnetic cell sorting using a murine monoclonal antibody against the melanoma-associated chondroitin sulfate proteoglycan. To prove the malignant origin of the positive cells and to define their chromosomal aberrations, we analyzed the genomes of 15 individually isolated cells from seven patients by single-cell comparative genomic hybridization (SCOMP).Results: Absolute and relative frequencies of circulating melanoma cells were associated with stage and with the presence or absence of detectable tumor. The detection of two or more cells correlated significantly with a reduced survival of patients with metastatic melanoma. All of the cells that were analyzed by SCOMP displayed multiple chromosomal changes and carried aberrations typical for melanoma.Conclusions: Immunomagnetic enrichment enables isolation and genomic characterization of circulating melanoma cells. The prognostic impact on survival of metastatic patients apparently reflects the aggressiveness of an ongoing tumor spread. Direct genomic analysis of the enriched and isolated cells will help to clarify the molecular-genetic basis of the establishment of generalized melanoma.

Mouse models indicate that metastatic dissemination occurs extremely early; however, the timing in human cancers is unknown. We therefore determined the time point of metastatic seeding relative to tumour thickness and genomic alterations in melanoma. Here, we find that lymphatic dissemination occurs shortly after dermal invasion of the primary lesion at a median thickness of ~0.5 mm and that typical driver changes, including BRAF mutation and gained or lost regions comprising genes like MET or CDKNA2, are acquired within the lymph node at the time of colony formation. These changes define a colonisation signature that was linked to xenograft formation in immunodeficient mice and death from melanoma. Thus, melanoma cells leave primary tumours early and evolve at different sites in parallel. We propose a model of metastatic melanoma dormancy, evolution and colonisation that will inform direct monitoring of adjuvant therapy targets.

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Purpose: In patients with uveal melanoma, tumor cell dissemination and subsequent formation of metastases are confined mainly to the hematogenous route. Here, we sought to isolate circulating melanoma cells in peripheral blood of patients with primary uveal melanoma and clinically localized disease. Experimental Design: Blood samples from 52 patients with clinically localized uveal melanoma and from 20 control individuals were prospectively collected before therapy of the primary tumor. Tumor cells expressing the melanoma-associated chondroitin sulfate proteoglycan were enriched by immunomagnetic cell sorting and visualized by immunocytologic staining. Results were compared with clinical data at presentation. Uveal melanoma is the most common primary intraocular malignant tumor in adults and 98% of these patients present with clinically localized disease (1, 2). Despite successful local tumor control, one half of the patients with primary uveal melanoma will die from metastatic disease (3). The 10-year survival rates in patients with nonmetastatic tumors range from 81% (T 1 N 0 M 0 ) to 15% (T 4 N 0 M 0 ; ref. 4). Metastasis-related deaths occur as long as 35 years after diagnosis, indicating that disseminated tumor cells may stay quiescent for decades and change their biological behavior even after this time (5). As therapeutic options in metastatic disease are poor, with a mean survival of 12 to 14 months (6, 7), there is an urgent need for early identification of patients at increased risk for metastases. This will allow evaluation of adjuvant therapeutic strategies in high-risk patients.Established prognostic factors for clinically localized ocular disease include largest basal tumor diameter (LBD), ciliary body involvement, and extraocular growth (8 -17). A number of staging classifications use combinations of the abovementioned criteria (for review, see ref. 4). However, neither of these factors alone or in combination is adequate for predicting the occurrence of metastatic disease in an individual patient. Other prognostic factors such as histologic subtype (11 -14, 16, 18) as well as genomic changes (19 -21) of the primary tumor also correlate with clinical outcome. Yet, these factors are increasingly difficult to determine as patients are currently often treated with radiotherapy. Although a diagnostic biopsy may be done before treatment, in most cases tumor tissue is not available for analysis.Metastatic spread in uveal melanoma is confined to the hematogenous route as long as the conjunctiva is not infiltrated trans-sclerally (22 blood is an obligate (although not sufficient) event in the metastatic cascade. Detection of circulating tumor cells might therefore represent a unique tool to identify patients at increased risk for metastatic disease. A recent systematic metaanalysis (23) of the prognostic value of tumor cell detection in peripheral blood in melanoma patients identified and evaluated two major approaches, PCR-based detection of melanomaassociated mRNA (n = 52 studies) and our rece...