This report presents for the first time, to our knowledge, cell lines characterized by microarray and grown from choroidal melanoma in patients who developed clinical metastases. Cell line characterization at passage 3 with 250K Mapping Arrays showed that each of the three cell lines and the two respective primary melanomas had common cytogenetic characteristics for monosomy 3, 6q loss, 8p loss, multiple 8q gain, and 16q loss. Two of the cell lines, MEL20–06–045 and MEL20–07–070 also demonstrated gains in chromosome 6p. Analysis of gene expression with U133 Plus 2.0 Arrays showed that two of the cell lines expressed gene characteristics similar to the respective primary melanoma for the 25 most overexpressed genes and the 20 most under-expressed genes in a comparative gene list composed of genes most overexpressed and under-expressed in an integrated analysis of choroidal melanoma with chromosome 3 loss versus 6p gain in the absence of chromosome 3 loss [11
]; the third cell line had a profile of gene expression similar to the two primary melanomas and the other two cell lines. Potentially relevant functional classes of biologic activity include G-protein coupled signaling, calcium response pathways, cell adhesion marker expression, retinoic acid response pathways and regulation of palmitoylation [11
Furthermore, sequence analysis from exon 5 of GNAQ
demonstrated heterozygous mutation of codon 209 in two of the three cell lines and the wild-type sequence in the third. GNAQ
codon 209 mutations in choroidal melanoma have been shown to be important in primary melanoma tumor development [14
Choroidal melanoma cell lines derived from melanomas that progressed to clinical metastases and patient death with cytogenetic characteristics and gene expression profiles closely corresponding to the respective primary melanoma are important for at least two reasons. First, there is no animal model of spontaneous uveal melanoma that accurately replicates the course of the human disease [16
]. Transgenic animal models of human disease do not reproduce the histiogenesis of human melanoma and animal melanoma differs substantially from human uveal melanoma [17
Second, cancer evolves through a process of step-wise accumulation of genetic alterations that result in uncontrolled cell proliferation and a lack of response to normal apoptotic stimuli. Genetic alterations include deletions, duplications/amplifications, translocations and point mutations that cause loss-of-function or gain-in-function and lead to altered protein expression. Availability of cell lines that closely correspond to the cells of metastasis-causing choroidal melanomas may serve as important in vitro models for the study of melanoma biology [19
Characterization of the choroidal melanoma cell lines in this report extends the initial attempt at uveal melanoma cell culture by Kirby [20
] in 1929, the report of six continuous cell lines from choroidal and ciliary body melanomas by Albert et al. [21
]. in 1984 and subsequent reports of cell lines derived from uveal melanomas [22
]. Cell lines from primary ciliary body and choroidal melanomas have been characterized by cellular morphology, relative growth rate, vasculogenic cord formation, immunohistochemistry, major histocompatability complex (MHC), and cytogenetic analysis of cell lines with karyotype study and determination of monosomy 3 status by microsatellite analysis. Cell lines derived from ciliary body and choroidal melanoma metastases have been studied by Luyton et al. [25
] using similar methods including karyotype analysis. Adding an additional dimension, this report presents analysis of primary choroidal melanomas and their cell lines with high resolution microarrays to compare cytogenetic characteristics and gene expression profiles.
Recognition that cancer is fundamentally a genetic disease emphasizes the importance of precisely characterizing the cytogenetic and gene expression of primary choroidal melanomas and the corresponding cell lines. Choroidal melanoma cell lines with genetic alterations that reflect their primary tumor provide malleable in vitro models. These models enable mechanistic dissection of melanoma biology, identification of “driver” genes and insight regarding therapeutic targets [29
Strengths of the three cell lines described in this report for in vitro study of choroidal melanoma biology and testing of therapeutic targets relate to (1) origin from primary choroidal melanomas that developed clinical evidence of metastasis <1.5 years after treatment of the primary tumor, (2) demonstration of cellular propagation through at least 6 passages, (3) documentation of equivalent cytogenetic aberrations and similar gene expression patterns in the primary melanomas and in the cell lines at passage 3, and (4) preservation of cell lines by freezing at passage 3 to provide highly characterized cell lines for future studies.
Limitations of the three cell lines as surrogates for study of melanoma biology, metastasis and therapy relate to (1) the limited but potentially significant differences in chromosomal aberration and gene expression between primary melanomas and corresponding cell lines, (2) the likelihood that, with long-term propagation and multiple passages, cell lines will undergo culture-related evolution in both chromosomal structure and gene expression, and (3) recognition that cancer biology is influenced by the in vivo micro-environment that is not replicated with in vitro models. However, the fact that the cell lines differ slightly between themselves may not be as important as having a diverse genetic background under which therapeutic drugs may be tested to determine biologic pathways in tumor development.
In summary, FNAB of primary choroidal melanomas that developed clinical metastases resulted in the development of three low-passage, highly characterized cell lines that exhibited the chromosomal aberrations and gene expression profiles present in the primary choroidal melanomas. These cell lines, with portions frozen beginning at passage 3 and portions continuing to propagate, provide in vitro models for study of choroidal melanoma biology, metastasis and therapy.