Visualization of Circulating Melanoma Cells in Peripheral Blood of Patients with Primary Uveal Melanoma

Abstract

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...

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

“…Based on the validated HMW-MAA antigen detection, 22,23 uveal melanoma CTC count was lower than in most epithelial tumor types, but was in line with previous results obtained in metastatic cutaneous melanoma patients. 18 Our results showed that elevated CTC count, using either a 1 or 2 CTC=7.5 mL positivity threshold, is associated with survival in our prospective Figure 1.…”

“…Of note, previous reports on blood CTC and bone marrow DTCs relying on highly similar detection approaches were reported in nonmetastatic uveal melanoma, with no major prognostic impact. 23,29,30 On the other hand, the use of ctDNA was made possible by the fact that most uveal melanomas harbor specific gene mutations in GNAQ and GNA11 genes. 3,4 Interestingly, these mutations are considered as being driver mutations and are thought to be stable over time in uveal melanoma cells.…”

Circulating Melanoma Cells

“…From these results it is obvious that; a) CTCs are present at relatively low concentrations; one tumour cell per 10 6 to 10 7 normal blood cells or on average, 1 cell per ml of blood 69,70,71 ; b) the number of cells appears to be related to stage 2,25,72 ; c) melanoma cell markers differ with respect to stage 73 ; and c) CTC gene expression differs from that of the primary tumour 28,29 . Quantitative RT-PCR has typically detected expression of melanocytic genes such as tyrosinase (TYR) 74,75 since normal melanocytes are not thought to circulate in peripheral blood and therefore detection of transcripts from melanocytic genes should correlate to identification of CTCs 72,76 .…”

“…Various methods have been used to quantify and characterise CTCs, including indirect methods, namely qRT-PCR 25,[62][63][64][65] , and direct analyses such as immunomagnetic bead capture, or fibre-optic array scanning technology 28,29,66,67,68 . From these results it is obvious that; a) CTCs are present at relatively low concentrations; one tumour cell per 10 6 to 10 7 normal blood cells or on average, 1 cell per ml of blood 69,70,71 ; b) the number of cells appears to be related to stage 2,25,72 ; c) melanoma cell markers differ with respect to stage 73 ; and c) CTC gene expression differs from that of the primary tumour 28,29 .…”

“…Several studies have shown that the number of CTCs in patient peripheral blood increases with increasing stages of melanoma 25,[26][27][28][29]30 . Yet the variety and phenotype of these CTCs, their ability to remain in circulation for many years thus evading the immune system, and their activation causing metastasis, remain largely unexplored, particularly for melanoma.…”