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Zuidervaart, W. (2005, May 25). Genomic and proteomic analysis in uveal melanoma. Retrieved from https://hdl.handle.net/1887/2696
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LINICAL ASPECTS OF UVEAL MELANOMAEpidemiology
Uveal melanoma is the most common primary intraocular tumor in adults in the Western world and represents 3% of all melanoma (Egan et al., 1988; Singh et al., 2003). The age-adjusted incidence of uveal melanoma varies from 5-8 patients per 1 million per year and has remained stable over the past 25 years (Singh et al., 2003).Uveal melanoma spread almost without any exception hematogenously, predominantly to the liver. The tumor is of an aggressive type: almost 50% of the patients with uveal melanoma will ultimately die from
metastatic disease (Jensen et al., 1982; McLean et al., 1993), while no effective treatment for metastases is yet available. Once the primary tumor has been diagnosed in the patient, the median survival time is 6.5 years (McLean et al., 1993). Life expectancy after metastases have been detected is only 2-9 months (Seddon et al., 1983; Albert et al., 1992; Kath et al., 1993).
Most uveal melanomas are located in the ciliary body and/or the choroid (23% and 72%,
respectively), whereas iris melanomas account for only a small percentage of these tumors, i.e. 5% (Yanoff and Fine, 1989). Iris melanomas are less aggressive with a low incidence of
developing metastases and are therefore not a subject of study in this thesis.
At the time of diagnosis, less than 2% of the patients have clinically detectable metastases, but
many patients may have already subclinical metastases (Donoso et al., 1985). It may be that treatment at this stage with e.g. immunotherapy may be the only way to improve survival in these patients, but would only be used in very high risk individuals. This highlights the critical need to identify prognostic markers indicative of uveal melanoma invasive and metastatic potential.
Diagnosis and treatment
Like cutaneous melanoma, uveal melanoma is originally derived, from melanocytes of the neurectoderm (choroid and ciliary body, Figure 1.1). Two main cell types have been recognized, i.e. the spindle cell type and the epithelioid cell type. Since both cell types are present in many uveal melanomas, additionally, the mixed cell type classification has been introduced (Zimmerman 1986).
Figure 1.1 Schematic view of an eye. (A) Choroid and (B) Ciliary body
In order to reduce the risk to develop metastases it is essential to treat the primary tumor as effectively as possible. For large tumors (Largest Tumor Diameter (LTD) > 15 mm and/or
thickness > 5 mm), enucleation remains the first choice of treatment, as well as in case of
extensive involvement of the optic disc or extensive extrascleral extension (Shields et al., 1991; Seregard and Kock 1995). For small (LTD < 10 mm and thickness ≤ 3 mm) and
medium-sized (LTD between 10-15 mm and/or thickness between 3-5 mm) tumors, different treatment strategies are used (Shields et al., 1991). These include local resection of the tumor (Damato 1993), plaque radiotherapy (Lommatzsch and Kirsch 1988; Shields and Shields., 1993; Lommatzsch et al., 2000), stereotactic radiotherapy (Zehetmayer et al., 2000) and thermotherapy (Oosterhuis et al., 1995). A new trend in ocular oncology is combining different treatment modalities such as transpupillary thermotherapy (TTT) combined with plaque radiotherapy to obtain better tumor control (Journee-de Korver et al., 1997; Seregard and Landau 2001).
Predisposing factors
Several parameters that predispose to uveal melanoma have been described, including
phenotypic risk factors. Race is one of the most significant host factors, as uveal melanoma is about 150 times more common in Caucasian than in African individuals (Egan et al., 1988; Singh et al., 2003) and is less common in Asians (Kuo et al., 1982; Biswas et al., 2002). People with light-colored eyes appear to be at a higher risk than dark-eyed people (Gallagher et al., 1985; Tucker et al., 1985) and subjects with brown eyes seems to be more protected against sunlight compared to those with blue eyes (Tucker et al., 1985; Holly et al., 1990). Although there is ample evidence of sunlight exposure as a risk factor for cutaneous
melanoma (Elwood and Jopson 1997; Gilchrest et al., 1999), the evidence in uveal melanoma is contradictory (Dolin et al., 1994; Egan et al., 1988). In a report by Li et al. (2000), the distribution of tumor origin correlated with the dose distribution of solar radiation on the retinal hemisphere, but these findings contradicted another report, in which the distribution of tumors in the choroid was found to be randomly distributed (Schwartz et al., 1997). Although there is biologic evidence that ultraviolet radiation induces DNA damage in cutaneous
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Melanoma
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melanoma (Gilchrest et al., 1999), still, no conclusive judgement about the role of sunlight in uveal melanoma can be made (reviewed by Singh 2004).
Uveal melanoma usually occurs sporadically in the absence of obvious genetic predisposing factors. However, in some patients, there might be a genetic predisposition. Familial uveal melanoma most often (63%) affects first-degree relatives, but rarely affects more than two
persons in one family, and therefore it may be associated with a generalized inherited predisposition to cancer (Singh et al., 1996).
Furthermore, there are a few clinical conditions that can predispose to or are associated with uveal melanoma, including ocular melanocytosis, neurofibromatosis type I, and familial atypical multiple mole melanoma syndrome (FAMMM). Ocular melanocytosis is a congenital condition characterized by hyperpigmentation of the uveal tract, sclera and epissclera. When hyperpigmentation of periocular skin takes place along the trigeminal nerve distribution, it is known as oculodermal melanocytosis (Nevus of Ota) (Gonder et al., 1982). Approximately 1,4% of uveal melanoma patients have oculodermal melanocytosis (ODM), indicating that
ODM is about 35 times more common in the uveal melanoma population (Gonder et al., 1982). The lifetime risk to develop uveal melanoma is estimated to be one of about 400 ODM patients compared to one out of 13,000 in the general population (Singh et al., 1998).
Neurofibromatosis type 1 has also been linked to uveal melanoma. This combination of tumors serves to emphasize the common neuroectodermal origin of tumors in this autosomal dominant condition (Specht and Smith 1988; Antle et al., 1990). In the FAMMM or
Dysplastic Nevus Syndrome, increased numbers of cutaneous nevi, cutaneous melanoma, as well as conjunctival nevi and uveal melanoma have been reported. Patients with uveal melanoma are more at risk to develop dysplastic nevi and cutaneous melanoma (Bataille et al., 1995; Van Hees et al., 1998). The p16 gene (CDKN2A) which is localized on
chromosome 9p21, is inactivated in a significant number of sporadic cancers, including cutaneous melanoma. It has been demonstrated that approximately 50% of all FAMMM
families show linkage to this region (Goldstein et al., 1994; Gruis et al., 1995). Whereas
p16ink4a (CDKN2A) is the main target for inactivation in cutaneous melanoma, mutation
screening and deletion mapping in uveal melanoma did not reveal the same results (Merbs et al., 1999). However, Van der Velden et al. (2001) demonstrated that hypermethylation of
p16ink4a is the cause of inactivation of this gene in uveal melanoma, which interestingly was
found more frequently in tumors from patients who developed metastatic disease. Germline
BRCA2 gene mutations have also been described to have an association with ocular melanoma
(Easton et al., 1997; Sinilnikova et al., 1999): they occur in 3% of patients younger than 50
years (Scott et al., 2002).
Prognostic indicators, clinical, immunological and histopathological parameters
also considered to be significant factors. Other immunological parameters are also of prognostic relevance. The number of CD3+ (T-lymphocytes) and CD4+ lymphocytes are
positively correlated with HLA expression (De Waard-Siebinga et al., 1996). HLA phenotype alterations is a common feature in various tumors, including cutaneous and uveal melanoma. In uveal melanoma, lack of expression of HLA-A as well as HLA-B antigens was found to be correlated with better patient survival (Blom et al., 1997). Most tumors lack HLA-C and HLA-G as well, but the biological importance of this is not yet clear (Hurks et al., 2001). These findings may suggest that shedding of uveal melanoma micrometastases with downregulated HLA class I antigen expression into the systemic circulation may facilitate their removal and prevent the development of metastases. It also may suggest that Natural Killer-cell-based lysis may be more effective in destroying blood-borne uveal melanoma cells than T-cell-mediated cytotoxicity (Blom et al., 1997; Jager et al., 2002; Anastassiou et al., 2003).
The expression of Epidermal Growth Factor Receptor (EGFR) has been found to be correlated with death due to metastatic disease in uveal melanoma (Hurks et al., 2000), with another report concluding that tumor-associated macrophages can express this receptor (Scholes et al., 2001). However, Ma and Niederkorn (1998) reported specific EGFR expression on uveal melanoma cell lines, indicating that these findings may not be exclusive.
Differences in invasive behaviour between uveal melanomas could be one of the most important factors for their differences in clinical outcome. Molecular markers like integrins and the involvement of cadherin-catenin adhesion complexes in the invasive potential of uveal melanoma cells have been the subject of several studies and have provided more insight in these complicated processes (Elshaw et al., 2001; Woodward et al., 2002; Seftor et al., 2002; Conway et al., 2003).
Cytogenetic and genetic markers as prognostic indicators will be discussed in the next paragraph.
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ROGNOSTIC INDICATORS, (C
YTO)G
ENETIC PARAMETERSChromosomal aberrations
Cytogenetic and comparative genomic hybridization analysis have revealed the involvement of several chromosomal aberrations in uveal melanoma. Sisley et al. (1997) identified a correlation with abnormalities in chromosomes 3, 6 and 8 and prognostic outcome.
Monosomy 3 in uveal melanoma has been proven to be an important determinant to predict metastatic potential (Prescher et al., 1996) and gain of chromosomal arm 8q, which includes the C-Myc oncogene locus, has been found in 65% of uveal melanomas (Speicher et al., 1994;
The subsequent paragraphs describe the potential involvement of genes and genetic pathways in uveal melanoma.
AKT and MAPK pathways
Another potential role in uveal melanogenesis involved the PTEN gene. The PTEN gene encodes a dual specific phosphatase, which plays a major role in the inhibition of cell migration and the formation of focal adhesions (Tamura et al., 1998). PTEN counteracts phosphatidylinositol 3-OH kinase (PI3-kinases) functions, which are associated with cell growth and survival (Di Cristofano and Pandolfi 2000; Vazquez and Sellers 2000). Somatic mutations of the PTEN gene have been detected in about 40% of cutaneous melanoma and
loss of this gene contributes to tumor development (Guldberg et al., 1997, Stahl et al., 2003). Furthermore, it has been recently found that there is a potential cooperation between the tumor suppressor gene PTEN and two other components of the RAS signaling network, NRAS and BRAF, suggesting that the MAPK (RAS) and the AKT pathways (PTEN) are frequently activated in parallel in melanogenesis (Tsao et al., 2004) However, no PTEN mutations were detected in a panel of uveal melanoma cell lines and cytogenetic
abnormalities involving chromosome 10q23 were not observed, as described in chapter 2 (Naus et al., 2000). The influence of the MAPK pathway in uveal melanoma will be discussed later in this chapter.
Rb and p53 pathways
Mutational deregulation of the cell-cycle is a hallmark of tumorigenesis (Hanahan and Weinberg 2000). The protein product of the Retinoblastoma gene (Rb) plays a central role as inhibitor of cellular proliferation (Bartek et al., 1997). The Rb gene is frequently mutated in certain cancers such as retinoblastoma, osteosarcoma, and small-cell lung cancer (Friend et al., 1986; Harbour et al., 1988), but no Rb mutations have been reported in uveal melanoma. This stimulated research into alterations in components of the interconnecting signaling pathways of the Rb gene and the p53 transcription factor (reviewed in Sherr and McCormick 2002). P53 is the most commonly mutated tumor suppressor in human cancer (Harris and Hollstein 1993), but no evidence exists for p53 mutations in either cutaneous or uveal melanoma (Chana et al., 1999; Kishore et al., 1996; Florenes et al., 1994). However, there is now evidence that functional abnormalities in both the Rb and p53 pathways are playing a role (Brantley and Harbour 2000a). HDM2 (human homologue of murine double minute MDM2)) is an inhibitor of p53 that targets p53 for degradation (Haupt et al., 1997). Although high expression of HDM2 in uveal melanoma cells has been related to poor clinical outcome (Coupland et al., 2000) and blocking experiments of HDM2 have led to rapid onset of
Recently, the mitogen-activated protein kinase (MAPK) pathway, has been found to be of great importance in the development of melanocytic neoplasia (Cohen et al., 2002;
Satyamoorthy et al., 2003). Of three RAS genes found to be activated by mutation in human tumors, NRAS (neuroblastoma RAS viral (v-ras) oncogene homolog) is most commonly mutated in cutaneous melanomas (Van Elsas et al., 1996). In the active GTP-bound state, RAS activates a number of downstream signalling cascades involved in controlling cell growth and behaviour. Initially, RAS interacts with and activates the serine/threonine protein kinase BRAF that acts in the MAPK pathway to transduce regulatory signals from RAS to MEK1/2. The signal tranducer mitogen-activated protein kinase/extracellular signal-related kinase kinase (MEK1/2) phosphorylates extracellular signal-regulated kinase (ERK1/2, p44/42), leading to the activation of these kinases, which in turn activate a variety of transcription factors, including ELK1, again through phosphorylation.
It has emerged that BRAF (v-raf murine sarcoma viral oncogene homolog B1) is very frequently activated by mutation in cutaneous nevi and melanomas (Davies et al., 2002; Pollock et al., 2003; Omholt et al., 2003; Cohen et al., 2004;Tsao et al., 2004). All BRAF mutations in cutaneous pigmented neoplasms occur within the kinase domain and most frequently consists of a 1796T A transversion in exon 15 (V599E) (Davies et al., 2002). Since cutaneous and uveal melanoma both arise from neural crest-derived melanocytes, the MAPK pathway came also to the attention in uveal melanoma research (see table III in chapter 3). Our data support the notion that activation of MAPK is indeed involved in development of uveal melanoma, but occurs via a different mechanism(s) to that in the majority of cutaneous melanomas (see chapter 3 in this thesis).
Genomics and Proteomics
The term genomics refers to the study of the human genome and proteomics refers to the analysis of the protein complement of the genome. With the introduction of high-throughput gene expression profiling techniques, and the enormous improvement in protein analysis technology, a major progress in the understanding of the involvement of multiple genes and gene products in association with uveal melanoma development, and cancer in general, have been established (Bittner et al., 2000 ; Tschentscher et al., 2003; Van der Velden et al., 2003; Zuidervaart et al., 2003).
This section describes the underlying methodology, as well as the present use of genomic and proteomic analysis, with special attention to its application in uveal melanoma research. Large scale gene expression analysis has proved to be an important strategy to identify gene
expression profiles, which are useful in classifying tissues according to pathological or prognostic subgroups. The unique quality of this technique lies in the ability to define
coordinate expression patterns, capable of representing a large number of relevant genes and a high degree of interaction, leading eventually to the detection of a potentially affected
expression data that may lead to a better understanding of the regulatory events involved in developmental processes in uveal melanoma.
G
ENOMICSGENE EXPRESSION PROFILING
Techniques
Different types of microarrays have been developed. One of the mostly used arrays are the gene expression arrays with thousands of different gene transcripts hybridized on nylon filters, glass slides or chips. Hybridization of tumor RNA on the carrier will provide a profile of genes that are transcribed in the tumor. Two types of arrays are frequently used, the cDNA-array (see Figure 1.2) and the oligonucleotide-cDNA-array (Affymetrix). Oligonucleotide-cDNA-arrays determine the expression of a messenger by measuring hybridization to a perfect match oligo and an internal control mismatch oligo (Lockhart et al., 1996).
Another microarray application is the tissue microarray (TMA) (Kononen et al., 1998). With this method, sections of multiple small tissue cores are placed on a glass slide.
Immunohistochemistry and In Situ Hybridisation can be performed on these sections revealing a clear impression of the tumor under study.
Gene expression profiling in uveal melanoma research
Maniotis et al. (1999) used cDNA microarray analysis to investigate the relationship between aggressive melanoma cell phenotype and the mechanism responsible for the generation of patterned matrix-associated vascular channels characteristic for both aggressive uveal melanoma and cutaneous melanoma. They furthermore used cDNA microarray analysis to confirm a genetic reversion to a pluripotent embryonic-like gene expression phenotype in highly aggressive uveal melanoma cells.
Seftor and coworkers (2002), extended their previous observations of vasculogenic mimicry by comparing gene expression profiles of highly invasive versus poorly invasive uveal melanoma cells from a heterogeneous tumor comprised of cells that had metastasized from the original tumor to the liver.
Figure 1.2 Flowchart of a microarray experiment.
P
ROTEOMICSTechniques
Two dimensional Poly-Acrylamide Gel Electrophoresis (2D-PAGE) is a powerful technique for protein separation, first according to pH (isoelectric point) and then acccording to size (molecular weight). The proteins can be visualized by staining with different agents, such as colloidal silver, fluorescent stains, or colloidal Coomassie Brilliant Blue G. Between 500-2000 spots can be reliably detected on large gels. Spots of interest are excized from the gel, digested, and analysed with Mass Spectrometry (MS). Proteins or peptides are ionized by electrospray ionization from liquid state (Fenn et al., 1989) or matrix-assisted laser desorption ionization (MALDI) from solid state (Karas and Hillenkamp 1988), and the mass of the ions is measured by various coupled analyzers (James 1997). The masses of the peptides can then be measured by MS, to produce a peptide mass fingerprint. This fingerprint will be compared with databases in order to identify the protein of interest (Figure 1.3). Protein microarrays have been developed recently as well, based on affinity (Haab et al., 2001; Schweitzer 2002). A great advantage is the decrease amount of material required, compared to the 2D method.
Test Reference RNA isolation Reverse transcriptase Labeling Cy5 (Red) R R R Cy3 (Green) G G G
Co-hybridization to microarray slide
Figure 1.3 Flowchart of a 2D-PAGE experiment.
Proteomics in cancer and uveal melanoma research
Proteomics is a powerful screening method for alterations in protein expression and posttranslational modifications. 2D-PAGE analysis in tumor research in general has been succesful in identifying proteins differentially expressed between normal liver cells and hepatocellular carcinoma cells (Yu et al., 2000) and in the identification of markers that differentiate between stages of bladder squamous cell carcinomas (Ostergaard et al., 1997). Although several examples of the use of proteomic research by 2D-PAGE have been
described for cutaneous melanoma (Bernard et al., 2003), the study by Missotten et al. (2003) is to our knowledge the only published project on proteomic analysis of uveal melanoma and outlines the use of proteomics in aqueous humor of uveal melanoma patients compared to a control group.
In chapter 8, two-dimensional electrophoresis (2D-PAGE) and mass spectrometry on a primary uveal melanoma cell line and two of its metastatic cell lines is used, in order to identify proteins associated with metastasis of uveal melanoma.
A
IMS AND SCOPE OF THIS THESISThe prognosis of patients with uveal melanoma is poor. Therefore, the importance to identify indicators of metastatic potential of uveal melanomas is essential. First of all, biological mechanisms that determine the level of aggressiveness of uveal melanoma are still unclear and secondly development of metastases is a complex mechanism in which many interrelated, but yet to identify, steps are involved.
Gene expression profiling technology has improved tremendously during the last couple of years. It is only a couple of years ago that expression profiling by microarray analysis has gained interest in human cancer research. 5 years ago conventional profiling allowed to study only a few hundreds of genes, whereas only a few years later, technology evolved in more affordable, and very advanced expression profiling techniques, providing data on more than
Condition A Condition B
Protein extraction
First dimension IEF
Second dimension SDS-PAGE
2DE image analysis Excision protein spots
and protein digestion MS analysis
Protein identification and characterization
30.000 genes. The same technological improvement has taken place in the analysis of the proteome. By taking advantage of these recent technological in genomic and proteomic research, the main outline of this thesis is to define prognostic markers and/or profiles to categorize uveal melanoma in groups in order to identify the biological mechanism of metastatic disease of uveal melanomas.
D
ESCRIPTION CONTENTS PER CHAPTERIn contrast to cutaneous melanoma, genetic mutations and biological evidence of aberrations in signalling pathways are less well described in uveal melanoma. Involvement of PTEN and the MAPK pathway in uveal melanoma, both proven relevant in cutaneous melanoma, are investigated in chapters 2 and 3. We assess whether the MAPK pathway is similarly
activated in melanoma of the uvea and screen for activating mutations in the NRAS, HRAS,
KRAS and BRAF genes in uveal melanoma cell lines and in primary uveal melanomas. The
expression level of the downstream MAPK pathway members MEK, ERK and ELK are determined to reveal any additional potential alterations.
In the past recent years, cDNA microarray studies have become a powerful tool to detect differences in gene expression in different types of cancer and subsequently demonstrated that expression profiling can serve as a prognostic tool. In chapters 4 and 5 of this thesis, filter
(macro) arrays are used with more than thousand cDNAs related to cancer development. To identify molecules involved in dissemination, expression profiles are compared using cell lines derived from a primary uveal melanoma and two liver metastases from the same patient as a model for tumor progression. In addition, to reveal genes suppressed by
hypermethylation, a comparison of expression profiles is performed between the primary melanoma cell line and the same cell line after demethylation by 5-aza-2'deoxycytidine treatment.
In chapter 5, uveal melanoma cell lines are compared to normal human melanocytes of the eye in order to reveal genes that are differentially expressed. Potential progression markers are subsequently validated for their prognostic value in primary uveal melanoma specimens. In the next two chapters gene expression profiling is put to a higher level by using 19K cDNA microarrays. In chapter 6, we use microarray expression profiling combined with phenotypic
characterization of uveal melanoma and melanocyte cell lines, in order to define a gene signature associated with invasive potential. In light of the observation that Wnt5a expression is associated with invasive potential of cutaneous melanoma, we determine in chapter 7 if
activation of the Wnt signalling pathway, through increased expression of Wnt5a, -catenin and MMP7, contributes to an invasive phenotype in uveal melanoma.
In chapter 8, we focus on the differential protein expression of a primary uveal melanoma
cell line and two of its metastatic cell lines, using two-dimensional electrophoresis (2D-PAGE) and mass spectrometry in order to identify proteins that are associated with metastasis of uveal melanoma.
In chapter 9 the previous studies and future research perspectives are discussed. To conclude,
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