Free
Anatomy and Pathology/Oncology  |   July 2011
Multiplex Ligation-Dependent Probe Amplification of Conjunctival Melanoma Reveals Common BRAF V600E Gene Mutation and Gene Copy Number Changes
Author Affiliations & Notes
  • Sarah L. Lake
    From the Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom; and
  • Fidan Jmor
    From the Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom; and
  • Justyna Dopierala
    From the Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom; and
  • Azzam F. G. Taktak
    the Department of Medical Physics and Clinical Engineering and
  • Sarah E. Coupland
    From the Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom; and
  • Bertil E. Damato
    the Liverpool Ocular Oncology Service, St. Paul's Eye Clinic, Royal Liverpool University Hospital, Liverpool, United Kingdom.
  • Corresponding author: Sarah L. Lake, Molecular and Clinical Cancer Medicine, Institute of Translational Medicine, University of Liverpool, Daulby Street, Liverpool, L69 3GA, UK; s.l.lake@liv.ac.uk
  • Footnotes
    2  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science July 2011, Vol.52, 5598-5604. doi:10.1167/iovs.10-6934
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Sarah L. Lake, Fidan Jmor, Justyna Dopierala, Azzam F. G. Taktak, Sarah E. Coupland, Bertil E. Damato; Multiplex Ligation-Dependent Probe Amplification of Conjunctival Melanoma Reveals Common BRAF V600E Gene Mutation and Gene Copy Number Changes. Invest. Ophthalmol. Vis. Sci. 2011;52(8):5598-5604. doi: 10.1167/iovs.10-6934.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: To determine the occurrence of BRAF V600E gene mutations and copy number changes of all autosome arms and genes known to be frequently altered in tumorigenesis in primary and metastatic conjunctival melanomas (CoMs).

Methods.: DNA (200 ng) was analyzed by three multiplex ligation-dependent probe amplification assays (P027 uveal melanoma, P036 human telomere, and P206 spitzoid melanoma).

Results.: Eight of 16 primary tumor samples and 4 of 6 metastatic samples showed BRAF V600E gene mutations. CDKN1A and RUNX2 (both 6p21.2) were amplified in 11 and 16 of 21 primary CoMs, respectively. In metastatic CoMs, MLH1 (3p22.1) and TIMP2 (17q25.3) were frequently amplified, and MGMT (20q26.3) and ECHS1 (10q26.3) were frequently deleted. The BDH (3q), FLJ20265 (4p), OPRL1 (20q), and PAO (10q) genes, representing the telomeres of their respective chromosome arms in the P036 assay, were frequently amplified in metastatic CoMs. No statistically significant associations were identified between BRAF mutation or CDKN1A or RUNX2 amplification and sex, age, histologic cell type, or patient survival.

Conclusions.: No copy number changes were associated exclusively with metastatic CoMs. However, further investigation of the role of CDKN1A and RUNX2 in CoMs development and that of MLH1, TIMP2, MGMT, and ECHS1 in metastatic CoMs is warranted. Validation of the observed gene and chromosome arm copy number changes in a larger cohort of primary and metastatic CoMs is necessary to identify the patients at highest risk for CoMs metastasis.

Conjunctival melanoma (CoMs) is a rare malignancy, representing approximately 1% to 5% of all ocular melanomas 1,2 and only 1.6% of all noncutaneous melanomas. 3 CoMs usually occurs in patients older than 60 years 4 and can arise in the palpebral, bulbar, or forniceal conjunctiva, plica semiluminaris, or caruncle. The etiology of CoMs is not well understood; however, some reports have suggested a possible role of increased exposure of the conjunctiva to ultraviolet light. 5 7 Approximately half of all CoMs are associated with conjunctival melanocytic intraepithelial neoplasia (C-MIN), 8 also termed primary acquired melanosis (PAM) with atypia, which has evolved into conjunctival melanoma in situ. 8,9 A small proportion are associated with nevi, whereas others appear to arise de novo. 10  
Local recurrence of CoMs occurs in >50% of patients, 4,6,11 and the 10-year mortality rate is 25% to 30%. 4,6,12 CoMs spread by lymphatics, most commonly to regional lymph nodes, but also hematogenously to the brain, liver, and lung. 13 The risk for death has been demonstrated to increase with melanoma de novo, caruncular location of CoMs, involvement of nonbulbar conjunctiva, local tumor recurrence, increased tumor thickness, high mitotic count, epithelioid cell morphology, and lymphatic invasion. 4,6,11,12,14  
Investigations of the genetic pathogenesis of CoMs have been limited mainly to BRAF mutational analysis and small cytogenetic studies. It has been suggested that CoMs may have a pathogenesis similar to that of cutaneous melanoma because of the presence of the BRAF V600E mutation, 15,16 commonly seen in these tumors. 17 The V600E mutation, however, has also been detected in conjunctival nevi, 18 which rarely transform into CoMs. 1,19 Other studies of the genetic changes in CoMs have suggested the presence of a variety of genetic changes, including loss of chromosomes 10q and 16q, 20 ; gains of RREB1 (6p25), cyclin D1 (11q 13), 21 and 4q 22 ; and polyploidy. 23 In the study by Busam et al., 21 relative gains of RREB1 and cyclin D1 were suggested to help distinguish between CoMs and conjunctival nevi. 
Although CoMs are histopathologically and clinically distinct from their more common intraocular counterpart, uveal melanoma (UM), 1 some studies have grouped all ocular melanomas together 20,24 or have investigated whether UM and CoMs share molecular genetic alterations. To date, the common gross chromosomal abnormalities seen in UM, including monosomy 3, polysomy 8q, 25 and GNAQ mutations, have not been detected in CoMs. 23,26 In contrast to CoMs, no BRAF mutations have been detected in UM. 15  
Prognostic genetic testing of uveal melanomas is becoming routine in a growing number of centers, because chromosome 3 loss and 8q gain are strong predictors of metastatic death. 27 31 Such testing has proved beneficial to patient well-being 32 and aids further patient management, such as by enabling screening for metastatic disease to be restricted to patients at high risk. 
The aims of this study were to determine, using multiplex ligation-dependent probe amplification (MLPA), whether frequent, nonrandom genetic changes also occurred in CoMs and whether these could potentially be used for prognostication purposes. 
Materials and Methods
Sample Selection
Thirty-six formalin-fixed, paraffin-embedded (FFPE) CoM tissue specimens were examined: these were from 33 patients, 13 of whom had been treated at the Charité University Hospital, Berlin, between 1994 and 2008 and 20 of whom had been treated at the Royal Liverpool and Broadgreen University Hospital, Liverpool, United Kingdom, between 1999 and 2009. Patients had been treated by varying methods, including excision, cryotherapy and/or topical mitomycin C. 
Twenty-nine samples were primary CoMs. Seven samples were metastatic CoMs excised from the parotid gland (n = 2), submandibular lymph node (n = 2), cervical lymph node (n = 1), nasopharyngeal tonsil (n = 1), and intraperitoneal abdomen (n = 1). No matched primary and metastasis samples from the same patient were available. Informed consent was obtained from each patient, and the research was performed according to the tenets of the Declaration of Helsinki. Local ethics committee approval was obtained before the study commenced. Ethical approval was obtained for this study from the Local Research Ethics Committee (LREC number 014/103). 
DNA Extraction
Areas of greater than 90% CM cells were identified after examination of hematoxylin and eosin–stained 4-μm FFPE sections. These areas were microdissected from 5–10-, and 20-μm sections, and DNA extraction was performed as described in Lake et al., 33 using a modified DNA purification kit (DNeasy Blood and Tissue kit; Qiagen, Crawley, UK). In brief, tissues were lysed in Proteinase K buffer (1.6–0.8 mg/mL proteinase K, 50 mM Tris, pH 8.5, 0.1 M NaCl, 1 mM EDTA, pH 8.0, 0.5% Tween-20, 0.5% NP40, 20 mM dithiothreitol) for 16 hours at 56°C, followed by a further 24 hours at 37°C. The DNA purification protocol was modified to include two washes with AW1 buffer, and DNA was eluted in 50 μL AE buffer. DNA quantity and A260/280 ratio were assessed with a spectrophotometer (NanoDrop; Thermo-Fisher Scientific, Cambridge, UK). 
Quality Control PCR
Multiplex PCR, adapted from the technique of van Dongen et al., 34 was performed to ensure sufficient DNA quality for analyses using 100 ng DNA; 25-μL reactions contained 1× high-performance buffer, 2 mM MgCl2, 0.8 mM dNTP mix, 0.625 U polymerase (ThermoStart; ABgene, Epsom, UK), 0.5% BSA (Sigma-Aldrich Company Ltd., Gillingham, UK), 0.1 μM forward and reverse primers for RAG1, PLZF, and AF4 exon 11, and 0.2 μM forward and reverse primers for AF4 exon 3 (Eurofins MWG Operon, London, UK). Reactions were performed using a thermal cycler (TC-412; Techne, Staffordshire, UK). PCR products were visualized on 2% agarose gels stained with 1× SYBR Safe (Invitrogen, Paisley, UK) with an imaging system (Bio Doc-It; Ultra-Violet Products Ltd., Cambridge, UK). 
Multiplex Ligation-Dependent Probe Amplification
MLPA was performed according to manufacturers' instructions (MRC-Holland, Amsterdam, The Netherlands) with the exception of the addition of 0.5% BSA (Sigma-Aldrich Company Ltd.) to the PCR step of the protocol. Experimental design and fragment analysis for the P027, P206, and P036 assays was as described in Damato et al. 19 In brief, six nontumor controls (FFPE tonsil tissue) were used in each MLPA assay with 200 ng DNA analyzed for both tumor and nontumor samples. MLPA reactions were performed in triplicate with a thermal cycler (G-Storm GS1; Gene Technologies Ltd., Essex, UK), and fragment detection was performed with a genetic analyzer (3130 Genetic Analyzer; Applied Biosystems, Paisley, UK). Peak heights were detected with genotype analysis software (GeneMarker; SoftGenetics, State College, PA) and were taken as a measure of intensity. Analysis of MLPA-PCR peak heights was based on the recommendations of the National Genetics Reference Laboratory (http://www.ngrl.org.uk/Manchester/publications) and was performed with computing software (MatLab R2009a; MathWorks, Natick, MA). 
Statistical Analysis
Statistical analyses were computed using SPSS (SPSS version 17.0; SPSS Science, Chicago, IL). Fisher's exact test was used to test the association between sex or tumor cell type and individual locus copy number changes. Age distributions in relation to individual locus copy number changes were evaluated using Mann-Whitney U testing. Survival analysis for samples based on observed copy number changes was conducted using log-rank testing after Kaplan-Meier analysis. 
Results
Patients and Samples
Thirty-six samples from 33 patients were analyzed by MLPA. Table 1 details the MLPA assays that were used to analyze each sample. Because of the small quantity of CoM tissue available, it was not possible to perform all three assays in some samples, leading to differing sample numbers being investigated for each assay. In summary, the following samples were analyzed: 21 patients with primary CoMs (n = 21 samples) and three patients with CoM metastasis (n = 4 samples) using the P027 assay; 28 patients with primary CoMs (n = 28 samples) and four patients with CoM metastasis (n = 7 samples) using the P036 assay; and 16 patients with primary CoMs (n = 16 samples) and three3 patients with CoM metastasis (n = 6 samples) by the P206 assay. Supplementary Table S1 details the samples analyzed by each MLPA assay. 
Table 1.
 
Clinicopathologic Features of CoM Patients
Table 1.
 
Clinicopathologic Features of CoM Patients
Patient Tumor Type Sex Age at Diagnosis (y) Histology Survival Cause of Death MLPA Analysis
Patient Outcome* Survival Time (mo) P027 P036 P206
1 P F 58 Spi Deceased 72 Metastatic melanoma
2 P F 61 Mix U U N/A
3 P M 82 Epi Survived 48 N/A
4 P F 55 Epi Survived 36 N/A
5 P M 80 Epi Survived 36 N/A
6 P F 54 Spi Deceased 2.5 Non-tumor related
7 P F 74 Spi Survived 12 N/A
8 P M 20 Spi Survived 12 N/A
9 P M 51 Mix Survived 12 N/A
10 P M 47 Mix Survived 7 N/A
11 P M 76 Mix Survived 12 N/A
12 P M 95 Epi Survived 12 N/A
13 P F 66 Mix Deceased 36 Metastatic melanoma
14 P M 66 Epi U U N/A
15 P M 59 Epi Survived 12 N/A
16 P M 46 Mix U U N/A
17 P F 84 Mix U U N/A
18 P M U Mix Deceased 48 Metastatic melanoma
19 P M 32 Mix Survived 108 N/A
20 P F 59 Mix Survived 96 N/A
21 P F 71 Mix Survived 84 N/A
22 P F 40 Mix Survived 72 N/A
23 P M 56 Mix Survived 60 N/A
24 P M 43 Mix Survived 60 N/A
25 P F 53 Mix Survived 48 N/A
26 P F 52 Mix Survived 48 N/A
27 P F 59 Spi Survived 36 N/A
28 P F 78 Epi Survived 36 N/A
29 P F 71 Spi Survived 84 N/A
30 Met F U Epi Deceased 12 U
31 Met F U Epi Deceased 48 Metastatic melanoma
32 Met F U Epi Survived 48 N/A
33 Met M U Mix U U N/A
Primary Conjunctival Tumors
The 29 patients were 14 men and 15 women with a median age at initial diagnosis of 59 years (range, 20–95 years). Histologic examination revealed six CoMs to be of spindle-cell type, 16 to be of mixed-cell type, and seven to be of epithelioid-cell type. Survival time for each patient was recorded from the date of diagnosis to December 2009. At the end of this study, four patients from this cohort had died of metastatic melanoma, with a median survival time of 42 months (range, 2.5–72 months). Survival details could not be obtained in four patients, who were lost to follow-up. 
Metastatic Conjunctival Tumors
Four patients (three women, one man) had metastatic CoMs. No information on age at initial diagnosis was available for these patients. Histologically, three of these tumors were of epithelioid-cell type and one was of a mixed-cell type. Two patients in this cohort died at the end of the study period, one from metastatic CoM. Table 1 illustrates the clinicopathologic features of all samples analyzed. 
Common Gene Copy Number Changes in Primary Conjunctival Melanomas and Metastases
Supplementary Table S1 details the gene copy number changes observed in each sample, across all loci tested by the three MLPA assays. An aberration was considered to be common if it occurred in >50% of samples. Only two such common aberrations—amplifications of the CDKN1A and RUNX2 genes (both 6p21.2)—were found in the primary CoMs analyzed: these were observed in 11 of 21 and 16 of 21 primary CoMs, respectively. Of the four metastases tested, amplifications of CDKN1A and RUNX2 were seen in three and four samples, respectively. Amplification of MLH1 (3p22.1) and TIMP2 (17q25.3) and deletion of MGMT and ECHS1 (both 10q26.3) were also frequently observed in the metastatic samples. Table 2 summarizes the aberrations that were observed using all three assays in the CoMs metastases. 
Table 2.
 
Common Genetic Aberrations in Metastatic CoMs
Table 2.
 
Common Genetic Aberrations in Metastatic CoMs
Gene Chromosomal Region Aberration No. Samples
MLH1 3p22.1 Amplification 3/4
RUNX2 6p21.2 Amplification 4/4
CDKN1A 6p21.2 Amplification 3/4
ECHS1 10q26.3 Deletion 4/6
MGMT 10q26.3 Deletion 5/6
TIMP2 17q25.3 Amplification 5/6
BDH 3q Amplification 6/7
FLJ20265 4p Amplification 6/7
PAO 10q Amplification 7/7
OPR1 20q Amplification 5/7
The P036 telomere assay identified amplification of the metastatic CoMs of the BDH, FLJ20265, and OPRL1 genes, corresponding to the subtelomeric regions of chromosome arms 3q, 4p, and 20q, respectively. All seven metastatic samples showed amplification of the PAO gene in the subtelomeric region of 10q. Conversely, only 2 of 28 primary CoMs analyzed by the P036 assay showed amplifications in the PAO gene. The BRAF V600E mutation was detected by the P206 assay in 8 of 16 primary CoMs and 4 of 6 metastatic samples (equating to two patients in this group). 
Statistics
Because of the small number of metastatic CoMs analyzed, the statistical calculations conducted were limited. Although copy number changes of CDKN1A and RUNX2 and the BRAF V600E mutation occurred in >50% of the primary CoMs, statistical analysis showed no significant association with clinicopathologic features such as sex, age, or cell type (Table 3). Kaplan-Meier analyses and log-rank testing, conducted to identify whether any of the common gene copy number changes or the V600E mutation were associated with patient survival, showed no statistically significant association either (Table 3). 
Table 3.
 
Clinicopathologic Features of Primary CoMs and Their Correlation with Amplification of CDKN1A and RUNX2
Table 3.
 
Clinicopathologic Features of Primary CoMs and Their Correlation with Amplification of CDKN1A and RUNX2
P027 Assay (n = 21)
With CDKN1A Amplification (n = 11) Without CDKN1A Amplification (n = 10) P With RUNX2 Amplification (n = 14) Without RUNX2 Amplification (n = 7) P
Sex 0.590* 0.562*
    Male 5 5 7 3
    Female 6 5 7 4
Age, y 0.654† 0.799†
    Median 66 56 59 55.5
    Range 20–95 40–76 20–95 40–74
Histology 0.857‡ 0.837‡
    Spindle 4 2 5 1
    Mixed 4 5 5 4
    Epithelioid 3 3 4 2
Survival at end of study period 0.356§ 0.329§
    Alive, n 10 5 11 4
    Dead, n 1 2 3 0
    Unknown, n 0 3 0 3
    Median, mo 36 36 36 48
    Range, mo 12–108 2.5–72 2.5–108 12–72
Discussion
To our knowledge, this is the first study to examine gene copy number changes at more than 120 loci and the presence of the BRAF V600E mutation in CoMs using MLPA. Statistical analysis was hampered by the paucity of CoM metastases available and the lack of paired primary and metastatic samples. Despite this, we have confirmed the presence of the BRAF V600E mutation and demonstrated frequent copy number alterations occurring in CoMs, particularly in metastatic lesions. Specifically, we have shown that CDKN1A and RUNX2 (both 6p21.2) were amplified in most primary CoMs. In metastatic CoMs, MLH1 (3p22.1) and TIMP2 (17q25.3) were frequently amplified, and MGMT (20q26.3) and ECHS1 (10q26.3) were frequently deleted. The BDH (3q), FLJ20265 (4p), OPRL1 (20q), and PAO (10q) genes, representing the telomeres of their respective chromosome arms in the P036 assay, were frequently amplified in metastatic CoMs. Because the detection of these aberrations occurred only in a small number of samples, the results of this study require future validation. 
Previous studies have investigated whether uveal and conjunctival melanomas share similar genetic alterations. 13,14 The common genetic aberrations occurring in uveal melanoma that are associated with patient mortality on chromosomes 1p, 3, 6, and 8 are detected by the P027 assay. 28 30,35 37 In using the P027 assay to analyze our cohort of CoM samples, we have confirmed that none of these gross chromosomal abnormalities are present in primary CoMs 23 or their metastases. Unlike uveal melanomas, which frequently show chromosome 3 loss, the metastatic CoM samples demonstrated frequent amplification of 3q. These data suggest a different molecular genetic pathogenesis for these two ocular malignancies. 19  
The P036 telomere assay is composed of probes in subtelomeric genes on all chromosome arms. These are not known to be involved in cancer. In using this assay, we assessed the copy number of these genes and used this information to infer potential alterations in copy number of the chromosomal arm on which the probe was located. It must be noted, however, that instability of the cancer genome could cause the genes tested to be amplified or deleted even if they were of no advantage to tumor development (also known as bystander aberrations); therefore, these results should be interpreted with some caution. The subtelomeric data presented requires validation using an alternative method, such as fluorescence in situ hybridization or comparative genomic hybridization, before definitive conclusions can be drawn regarding the gross chromosomal abnormalities common in metastatic CoMs. 
There have been few previous cytogenetic studies of CoMs, and those that have been conducted have been varied in their approaches and results and have used small sample sizes, probably because of the paucity of available tissue of this rare tumor type. 20,22,23,38 These studies are consistent in demonstrating a complex genotype in CoMs, in agreement with our observations of multiple genetic changes using MLPA. 20,22,23,38 There are, however, some discrepancies: for example, Vadjic et al. 20 reported losses of 10q genes that are discordant with our observation of 10q gains. The differences in the number of samples and the methodology may explain these differences. Although Vadjic et al. 20 applied a higher resolution technique (comparative genomic hybridization) than that used in the present study, they only analyzed two CoMs. Our observation of all seven CoMs metastases with a 10q amplification, and the commonness of amplification of ECHS1 and MGMT (10q26.3), indeed suggest that genes of importance for metastatic progression in CoMs occur in this region. 
Using MLPA and whole tumor sections, we were unable to examine for the presence of polyploidy in CoMs or polysomy 4q cell clones, as previously described by others. 22 23 In agreement with our findings in CoMs, however, three studies in cutaneous melanoma have reported common amplifications of 20q, suggesting this region also may harbor genes essential in the pathogenesis of melanoma. 39 41  
Our findings of frequent amplification of CDKN1A and RUNX2 in primary and metastatic CoM samples, in the absence of common amplification of 6p indicated by results from the P036 assay, implies that these genes may play significant roles in CoM development. For RUNX2, this notion is supported by studies in cutaneous melanoma 42 44 as well as in breast and prostate cancer, in which increased expression has previously been observed to enhance the motility and invasion of cells. 45,46 Further, in a mouse melanoma model, tumor size reduction and metastasis prevention was achieved when RUNX2 expression was promoted by a small molecule inhibitor of TGF-β signaling, SD-208. 47  
p21(CDKN1A)–mediated senescence has been implicated as a tumor suppressive mechanism in cutaneous melanoma, in a BRAF V600E-dependent manner. 48 Our observation of CDKN1A amplification in CoMs suggest that p21 may have another role in CoMs tumorigenesis. CDKN1A amplification, however, may be a nonfunctional biomarker of disease progression; further study of p21 at the mRNA and protein levels is, therefore, required to determine whether the role of this gene in CoMs is divergent from that proposed in cutaneous melanoma. 
The other genes found to be frequently amplified in the CM metastases, TIMP2 and MLH1, are of potential interest in CoMs because of their established role in other cancers. Because TIMP2 is an inhibitor of the matrix metalloproteinases that degrade the extracellular matrix, it could be supposed that its most likely role in tumorigenesis is that of a metastasis suppressor. However, differing roles for TIMP2 in metastasis have been reported, with some studies 49 51 suggesting that it promotes and others 52 suggesting that it hinders the metastatic process. This appears to depend on the tissue origin of the tumor. The amplification of TIMP2 in our present study suggests that TIMP2 promotes metastases in CoMs; however, we plan to validate this finding on a larger cohort of primary and metastatic CoMs. 
With respect to MLH1, the few studies addressing this gene in cancer have demonstrated microsatellite instability for the locus in colorectal cancer 53 and gene methylation in thyroid cancer. 54 These findings appear to contradict the amplification of MLH1 seen in the CoM metastases in the present study. We assessed, however, only gene dosage; epigenetic regulation may also play a part in ultimately controlling gene expression levels. Investigations of epigenetic deregulation in CoMs are yet to be described and, as with other cancer types, may provide useful insight into their tumorigenesis. 55 57  
The most frequent deletions we observed in the present study involved ECHS1 (10q26.3) and MGMT (20q26.3) in metastatic CoMs. To date, little is known about the role of the ECHS1 gene, encoding an Enoyl-CoA hydratase, in cancer. Deletion of MGMT in CoM metastases is consistent with the published literature. 58 Of particular relevance to CoMs are studies in uveal melanoma that have demonstrated a reduction in MGMT protein expression and activity in liver metastases compared with healthy liver tissue. 59 Because CoMs are known also to metastasize to the liver, decreased gene dosage of MGMT may be a potential mechanism facilitating the colonization of CoM cells in the liver. 
Eight of 16 primary tumor samples and 4 of 6 metastatic samples carried the BRAF V600E mutation, a marginally higher frequency than seen in previous studies of 14% to 40%. 15,16,18,20 In these latter investigations, BRAF V600E mutation was primarily detected or validated using direct sequencing. Because of the small sample sizes, sufficient DNA was not available in the present study to validate the MLPA data using direct sequencing. Our use of an alternative methodology may explain the higher than previously detected incidence of BRAF V600E mutation in CoMs. However, comparison of gene dosage detection by MLPA with other techniques, such as microsatellite analysis and fluorescence in situ hybridization, has determined MLPA to be a robust and sensitive method. 35,60 Therefore, our experience and that of others in successfully applying MLPA to detect gene dosage and tumor suppressor gene hypermethylation (Ma et al., manuscript in preparation) 25,35,61 63 suggest that the incidence of BRAF V600E mutation in primary and metastatic CoMs represents genuine differences in the cohorts analyzed. 
BRAF mutation has an overall occurrence of approximately 40% in cutaneous melanoma, 64 indicating a frequency of mutation similar to that detected in CoMs. The larger number of persons with cutaneous melanoma than with CoM has allowed powerful genetic studies to be performed to dissect the molecular pathogenesis of this disease, revealing, for example, the association of lymph node metastasis and higher AJCC stage for lesions with BRAF mutations. 65 67 Although BRAF mutation is thought to be an early event in the development of cutaneous melanoma, 68 it is not considered to be an initiating event or to be involved in familial disposition; instead, it is proposed to promote melanoma progression. 69,70 The hypothesis that exposure to UV is causative in BRAF mutation development is supported by the higher incidence of BRAF mutation in intermittently sun-exposed sites compared with sites with no sun exposure. 71 However, in the same study, BRAF mutation was also found to be less frequent in cutaneous melanomas at sites with chronic sun damage, suggesting different etiologies for cutaneous melanoma. From the comparable incidence of BRAF mutation in cutaneous and conjunctival melanoma, it can be hypothesized that intermittent UV exposure may play a role in CoM development and that, should larger cohorts of CoMs be examined, robust associations between gene mutation and disease progression may be identified. If BRAF mutation were to be demonstrated as key to CoM pathogenesis, trials of oncogene-targeted therapies, such as PLX4023, a drug that is undergoing phase III clinical trials in cutaneous melanoma, could prove beneficial. 72  
Although a few studies have investigated the presence of BRAF V600E mutations in CoMs, 15,18 only one study could demonstrate an association with clinicopathologic factors, namely, the presence of necrosis. 16 Given that these studies have also analyzed relatively small cohorts of CoMs 15,16,18,20 and that none have looked specifically at metastases, our current investigation is an important addition to the analysis of the genetics of CoMs. 
Future studies will focus on validating whether gains of the subtelomeric BDH, FLJ20265, PAO, and OPRL1 genes do indicate gains of chromosome arms 3q, 4p, 10q, and 20q. This is of particular importance for the 10q region, which showed amplification in all seven metastatic samples but only two primary CoMs. This alteration, along with those others observed in metastasis samples, is, therefore, of interest for future prognostic testing. 
Although no metastatic sample was accompanied by its original primary tumor, the difference in the frequency of genetic changes between the two groups is striking. Without more detailed survival data, it is impossible to determine whether patients with those primary tumors harboring amplifications more commonly seen in the metastases (e.g., PAO [10q] or TIMP2) will develop metastases in time. The aberrations detected, in particular those seen commonly in metastases, require further validation in a larger cohort of CoMs with more comprehensive clinical and follow-up information. Such a study would establish whether the genetic changes identified here are important factors in the pathology of CoMs and could be used to identify CoM patients at high risk for metastatic spread. Knowledge of these aberrations may improve understanding of the cell signaling pathways that are deregulated in CoMs and that ultimately improve patient treatment, particularly in patients with metastatic disease. 
Supplementary Materials
Table st1, XLS - Table st1, XLS 
Footnotes
 Presented at the annual meeting of the European Association for Eye and Vision Research, Crete, Greece, October 6–9, 2010.
Footnotes
 Supported by Fight for Sight Grant 1685 (SL), Northwest Cancer Research Fund (FJ), Eye Tumor Research Fund of the Royal Liverpool University Hospital (JD), National Health Service, UK (AT), and National Commissioning Group of the National Health Service, UK (BD, SC).
Footnotes
 Disclosure: S.L. Lake, None; F. Jmor, None; J. Dopierala, None; A.F.G. Taktak, None; S.E. Coupland, None; B.E. Damato, None
The authors thank Harald Stein (Department of Pathology, Charité, Campus Benjamin Franklin, Berlin, Germany) for providing conjunctival melanoma specimens and clinical data. 
References
Seregard S . Conjunctival melanoma. Surv Ophthalmol. 1998;42:321–350. [CrossRef] [PubMed]
Shields CL . Conjunctival melanoma. Br J Ophthalmol. 2002;86:127. [CrossRef] [PubMed]
Scotto J Fraumeni JFJr Lee JA . Melanomas of the eye and other noncutaneous sites: epidemiologic aspects. J Natl Cancer Inst. 1976;56:489–491. [PubMed]
Missotten GS Keijser S De Keizer RJ De Wolff-Rouendaal D . Conjunctival melanoma in the Netherlands: a nationwide study. Invest Ophthalmol Vis Sci. 2005;46:75–82. [CrossRef] [PubMed]
Tuomaala S Eskelin S Tarkkanen A Kivela T . Population-based assessment of clinical characteristics predicting outcome of conjunctival melanoma in whites. Invest Ophthalmol Vis Sci. 2002;43:3399–3408. [PubMed]
Shields CL . Conjunctival melanoma: risk factors for recurrence, exenteration, metastasis, and death in 150 consecutive patients. Trans Am Ophthalmol Soc. 2000;98:471–492. [PubMed]
Silvers D Jakobiec FA Freeman T . Melanoma of the conjunctiva: a clinicopathologic study. In: Jakobiec FA ed. Ocular and Adnexal Tumors. Birmingham, UK: Aesculapius; 1978:583–599.
Damato B Coupland SE . Clinical mapping of conjunctival melanomas. Br J Ophthalmol. 2008;92:1545–1549. [CrossRef] [PubMed]
Damato B Coupland SE . Conjunctival melanoma and melanosis: a reappraisal of terminology, classification and staging. Clin Exp Ophthalmol. 2008;36:786–795. [CrossRef]
de Wolff-Rouendaal D . Melanozytar̈e tumoren der bindehaut. In: Lommatzsch P ed. Ophthalmologische onkologie. Stuttgart, Germany: Enke; 1990:81–95.
Anastassiou G Heiligenhaus A Bechrakis N Bader E Bornfeld N Steuhl KP . Prognostic value of clinical and histopathological parameters in conjunctival melanomas: a retrospective study. Br J Ophthalmol. 2002;86:163–167. [CrossRef] [PubMed]
Desjardins L Poncet P Levy C Schlienger P Asselain B Validire P . [Prognostic factors in malignant melanoma of the conjunctiva: an anatomo-clinical study of 56 patients]. J Fr Ophtalmol. 1999;22:315–321. [PubMed]
Tuomaala S Kivela T . Metastatic pattern and survival in disseminated conjunctival melanoma: implications for sentinel lymph node biopsy. Ophthalmology. 2004;111:816–821. [CrossRef] [PubMed]
Damato B Coupland SE . An audit of conjunctival melanoma treatment in Liverpool. Eye (Lond). 2009;23:801–809. [CrossRef] [PubMed]
Spendlove HE Damato BE Humphreys J Barker KT Hiscott PS Houlston RS . BRAF mutations are detectable in conjunctival but not uveal melanomas. Melanoma Res. 2004;14:449–452. [CrossRef] [PubMed]
Gear H Williams H Kemp EG Roberts F . BRAF mutations in conjunctival melanoma. Invest Ophthalmol Vis Sci. 2004;45:2484–2488. [CrossRef] [PubMed]
Davies H Bignell GR Cox C . Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–954. [CrossRef] [PubMed]
Goldenberg-Cohen N Cohen Y Rosenbaum E . T1799A BRAF mutations in conjunctival melanocytic lesions. Invest Ophthalmol Vis Sci. 2005;46:3027–3030. [CrossRef] [PubMed]
Brownstein S . Malignant melanoma of the conjunctiva. Cancer Control. 2004;11:310–316. [PubMed]
Vajdic CM Hutchins AM Kricker A . Chromosomal gains and losses in ocular melanoma detected by comparative genomic hybridization in an Australian population-based study. Cancer Genet Cytogenet. 2003;144:12–17. [CrossRef] [PubMed]
Busam KJ Fang Y Jhanwar SC Pulitzer MP Marr B Abramson DH . Distinction of conjunctival melanocytic nevi from melanomas by fluorescence in situ hybridization. J Cutan Pathol. 2010;37:196–203. [CrossRef] [PubMed]
Dahlenfors R Tornqvist G Wettrell K Mark J . Cytogenetical observations in nine ocular malignant melanomas. Anticancer Res. 1993;13:1415–1420. [PubMed]
McNamara M Felix C Davison EV Fenton M Kennedy SM . Assessment of chromosome 3 copy number in ocular melanoma using fluorescence in situ hybridization. Cancer Genet Cytogenet. 1997;98:4–8. [CrossRef] [PubMed]
Sinilnikova OM Egan KM Quinn JL . Germline brca2 sequence variants in patients with ocular melanoma. Int J Cancer. 1999;82:325–328. [CrossRef] [PubMed]
Damato B Dopierala JA Coupland SE . Genotypic profiling of 452 choroidal melanomas with multiplex ligation-dependent probe amplification. Clin Cancer Res. 2010;16:6083–6092. [CrossRef] [PubMed]
Dratviman-Storobinsky O Cohen Y Frenkel S Pe'er J Goldenberg-Cohen N . Lack of oncogenic GNAQ mutations in melanocytic lesions of the conjunctiva as compared to uveal melanoma. Invest Ophthalmol Vis Sci. 2010;51:6180–6182. [CrossRef] [PubMed]
Damato B Duke C Coupland SE . Cytogenetics of uveal melanoma: a 7-year clinical experience. Ophthalmology. 2007;114:1925–1931. [CrossRef] [PubMed]
Prescher G Bornfeld N Hirche H Horsthemke B Jockel KH Becher R . Prognostic implications of monosomy 3 in uveal melanoma. Lancet. 1996;347:1222–1225. [CrossRef] [PubMed]
Mensink HW Kilic E Vaarwater J Douben H Paridaens D de Klein A . Molecular cytogenetic analysis of archival uveal melanoma with known clinical outcome. Cancer Genet Cytogenet. 2008;181:108–111. [CrossRef] [PubMed]
Kilic E Naus NC van Gils W . Concurrent loss of chromosome arm 1p and chromosome 3 predicts a decreased disease-free survival in uveal melanoma patients. Invest Ophthalmol Vis Sci. 2005;46:2253–2257. [CrossRef] [PubMed]
Damato B . Legacy of the collaborative ocular melanoma study. Arch Ophthalmol. 2007;125:966–968. [CrossRef] [PubMed]
Cook SA Damato B Marshall E Salmon P . Psychological aspects of cytogenetic testing of uveal melanoma: preliminary findings and directions for future research. Eye (Lond). 2009;23:581–585. [CrossRef] [PubMed]
Lake SL Coupland SE Taktak AF Damato BE . Whole-genome microarray detects deletions and loss of heterozygosity of chromosome 3 occurring exclusively in metastasizing uveal melanoma. Invest Ophthalmol Vis Sci. 2010;51:4884–4891. [CrossRef] [PubMed]
van Dongen JJ Langerak AW Bruggemann M . Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98–3936. Leukemia. 2003;17:2257–2317. [CrossRef] [PubMed]
Damato B Dopierala J Klaasen A van Dijk M Sibbring J Coupland SE . Multiplex ligation-dependent probe amplification of uveal melanoma: correlation with metastatic death. Invest Ophthalmol Vis Sci. 2009;50:3048–3055. [CrossRef] [PubMed]
Aalto Y Eriksson L Seregard S Larsson O Knuutila S . Concomitant loss of chromosome 3 and whole arm losses and gains of chromosome 1, 6, or 8 in metastasizing primary uveal melanoma. Invest Ophthalmol Vis Sci. 2001;42:313–317. [PubMed]
White VA Chambers JD Courtright PD Chang WY Horsman DE . Correlation of cytogenetic abnormalities with the outcome of patients with uveal melanoma. Cancer. 1998;83:354–359. [CrossRef] [PubMed]
Aubert C Rouge F Reillaudou M Metge P . Establishment and characterization of human ocular melanoma cell lines. Int J Cancer. 1993;54:784–792. [CrossRef] [PubMed]
Namiki T Yanagawa S Izumo T . Genomic alterations in primary cutaneous melanomas detected by metaphase comparative genomic hybridization with laser capture or manual microdissection: 6p gains may predict poor outcome. Cancer Genet Cytogenet. 2005;157:1–11. [CrossRef] [PubMed]
Pirker C Holzmann K Spiegl-Kreinecker S . Chromosomal imbalances in primary and metastatic melanomas: over-representation of essential telomerase genes. Melanoma Res. 2003;13:483–492. [CrossRef] [PubMed]
Koynova DK Jordanova ES Milev AD . Gene-specific fluorescence in-situ hybridization analysis on tissue microarray to refine the region of chromosome 20q amplification in melanoma. Melanoma Res. 2007;17:37–41. [CrossRef] [PubMed]
Packer LM Pavey SJ Boyle GM . Gene expression profiling in melanoma identifies novel downstream effectors of p14ARF. Int J Cancer. 2007;121:784–790. [CrossRef] [PubMed]
Jiang H Lin J Su ZZ . The melanoma differentiation-associated gene mda-6, which encodes the cyclin-dependent kinase inhibitor p21, is differentially expressed during growth, differentiation and progression in human melanoma cells. Oncogene. 1995;10:1855–1864. [PubMed]
Riminucci M Corsi A Peris K Fisher LW Chimenti S Bianco P . Coexpression of bone sialoprotein (BSP) and the pivotal transcriptional regulator of osteogenesis, Cbfa1/Runx2, in malignant melanoma. Calcif Tissue Int. 2003;73:281–289. [CrossRef] [PubMed]
Baniwal SK Khalid O Gabet Y . Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis. Mol Cancer. 2010;9:258. [CrossRef] [PubMed]
Leong DT Lim J Goh X . Cancer-related ectopic expression of the bone-related transcription factor RUNX2 in non-osseous metastatic tumor cells is linked to cell proliferation and motility. Breast Cancer Res. 2010;12:R89. [CrossRef] [PubMed]
Mohammad KS Javelaud D Fournier PG . TGF-beta-RI kinase inhibitor SD-208 reduces the development and progression of melanoma bone metastases. Cancer Res. 2011;71:175–184. [CrossRef] [PubMed]
de Keizer PL Packer LM Szypowska AA . Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence. Cancer Res. 2010;70:8526–8536. [CrossRef] [PubMed]
Park KS Kim SJ Kim KH Kim JC . Clinical characteristics of TIMP2, MMP2, and MMP9 gene polymorphisms in colorectal cancer. J Gastroenterol Hepatol. 2011;26:391–397. [CrossRef] [PubMed]
Staack A Badendieck S Schnorr D Loening SA Jung K . Combined determination of plasma MMP2, MMP9, and TIMP1 improves the non-invasive detection of transitional cell carcinoma of the bladder. BMC Urol. 2006;6:19. [CrossRef] [PubMed]
Gentner B Wein A Croner RS . Differences in the gene expression profile of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in primary colorectal tumors and their synchronous liver metastases. Anticancer Res. 2009;29:67–74. [PubMed]
Mendes O Kim HT Lungu G Stoica G . MMP2 role in breast cancer brain metastasis development and its regulation by TIMP2 and ERK1/2. Clin Exp Metastasis. 2007;24:341–351. [CrossRef] [PubMed]
Pancione M Forte N Fucci A . Prognostic role of beta-catenin and p53 expression in the metastatic progression of sporadic colorectal cancer. Hum Pathol. 2010;41:867–876. [CrossRef] [PubMed]
Guan H Ji M Hou P . Hypermethylation of the DNA mismatch repair gene hMLH1 and its association with lymph node metastasis and T1799A BRAF mutation in patients with papillary thyroid cancer. Cancer. 2008;113:247–255. [CrossRef] [PubMed]
Maat W Beiboer SH Jager MJ Luyten GP Gruis NA van der Velden PA . Epigenetic regulation identifies RASEF as a tumor-suppressor gene in uveal melanoma. Invest Ophthalmol Vis Sci. 2008;49:1291–1298. [CrossRef] [PubMed]
Onken MD Worley LA Harbour JW . A metastasis modifier locus on human chromosome 8p in uveal melanoma identified by integrative genomic analysis. Clin Cancer Res. 2008;14:3737–3745. [CrossRef] [PubMed]
Zeschnigk M Tschentscher F Lich C Brandt B Horsthemke B Lohmann DR . Methylation analysis of several tumour suppressor genes shows a low frequency of methylation of CDKN2A and RARB in uveal melanomas. Comp Funct Genomics. 2003;4:329–336. [CrossRef] [PubMed]
Myong NH . Role of Loss of O-methylguanine DNA methyltransferase (MGMT) expression in non-small cell lung carcinomas (NSCLCs): with reference to the relationship with p53 overexpression. Cancer Res Treat. 2010;42:95–100. [CrossRef] [PubMed]
Voelter V Diserens AC Moulin A . Infrequent promoter methylation of the MGMT gene in liver metastases from uveal melanoma. Int J Cancer. 2008;123:1215–1218. [CrossRef] [PubMed]
Funari MF Jorge AA Souza SC . Usefulness of MLPA in the detection of SHOX deletions. Eur J Med Genet. 2010;53:234–238. [CrossRef] [PubMed]
Cabello MJ Grau L Franco N . Multiplexed methylation profiles of tumor suppressor genes in bladder cancer. J Mol Diagn. 2011;13:29–40. [CrossRef] [PubMed]
Conway C Beswick S Elliott F . Deletion at chromosome arm 9p in relation to BRAF/NRAS mutations and prognostic significance for primary melanoma. Genes Chromosomes Cancer. 2011;49:425–438. [CrossRef]
Fabi A Di Benedetto A Metro G . HER2 protein and gene variation between primary and metastatic breast cancer: significance and impact on patient care. Clin Cancer Res. 2011;17:2055–2064. [CrossRef] [PubMed]
Lee JH Choi JW Kim YS . Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis. Br J Dermatol. 2011;164:776–7840. [CrossRef] [PubMed]
Ellerhorst JA Greene VR Ekmekcioglu S . Clinical correlates of NRAS and BRAF mutations in primary human melanoma. Clin Cancer Res. 2010;17:229–235. [CrossRef] [PubMed]
Goldstein NS . Serrated pathway and APC (conventional)-type colorectal polyps: molecular-morphologic correlations, genetic pathways, and implications for classification. Am J Clin Pathol. 2006;125:146–153. [CrossRef] [PubMed]
Broekaert SM Roy R Okamoto I . Genetic and morphologic features for melanoma classification. Pigment Cell Melanoma Res. 2010;23:763–770. [CrossRef] [PubMed]
Omholt K Platz A Kanter L Ringborg U Hansson J . NRAS and BRAF mutations arise early during melanoma pathogenesis and are preserved throughout tumor progression. Clin Cancer Res. 2003;9:6483–6488. [PubMed]
Laud K Kannengiesser C Avril MF . BRAF as a melanoma susceptibility candidate gene? Cancer Res. 2003;63:3061–3065. [PubMed]
Meyer P Klaes R Schmitt C Boettger MB Garbe C . Exclusion of BRAFV599E as a melanoma susceptibility mutation. Int J Cancer. 2003;106:78–80. [CrossRef] [PubMed]
Maldonado JL Fridlyand J Patel H . Determinants of BRAF mutations in primary melanomas. J Natl Cancer Inst. 2003;95:1878–1890. [CrossRef] [PubMed]
Flaherty KT Puzanov I Kim KB . Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010;363:809–819. [CrossRef] [PubMed]
Table 1.
 
Clinicopathologic Features of CoM Patients
Table 1.
 
Clinicopathologic Features of CoM Patients
Patient Tumor Type Sex Age at Diagnosis (y) Histology Survival Cause of Death MLPA Analysis
Patient Outcome* Survival Time (mo) P027 P036 P206
1 P F 58 Spi Deceased 72 Metastatic melanoma
2 P F 61 Mix U U N/A
3 P M 82 Epi Survived 48 N/A
4 P F 55 Epi Survived 36 N/A
5 P M 80 Epi Survived 36 N/A
6 P F 54 Spi Deceased 2.5 Non-tumor related
7 P F 74 Spi Survived 12 N/A
8 P M 20 Spi Survived 12 N/A
9 P M 51 Mix Survived 12 N/A
10 P M 47 Mix Survived 7 N/A
11 P M 76 Mix Survived 12 N/A
12 P M 95 Epi Survived 12 N/A
13 P F 66 Mix Deceased 36 Metastatic melanoma
14 P M 66 Epi U U N/A
15 P M 59 Epi Survived 12 N/A
16 P M 46 Mix U U N/A
17 P F 84 Mix U U N/A
18 P M U Mix Deceased 48 Metastatic melanoma
19 P M 32 Mix Survived 108 N/A
20 P F 59 Mix Survived 96 N/A
21 P F 71 Mix Survived 84 N/A
22 P F 40 Mix Survived 72 N/A
23 P M 56 Mix Survived 60 N/A
24 P M 43 Mix Survived 60 N/A
25 P F 53 Mix Survived 48 N/A
26 P F 52 Mix Survived 48 N/A
27 P F 59 Spi Survived 36 N/A
28 P F 78 Epi Survived 36 N/A
29 P F 71 Spi Survived 84 N/A
30 Met F U Epi Deceased 12 U
31 Met F U Epi Deceased 48 Metastatic melanoma
32 Met F U Epi Survived 48 N/A
33 Met M U Mix U U N/A
Table 2.
 
Common Genetic Aberrations in Metastatic CoMs
Table 2.
 
Common Genetic Aberrations in Metastatic CoMs
Gene Chromosomal Region Aberration No. Samples
MLH1 3p22.1 Amplification 3/4
RUNX2 6p21.2 Amplification 4/4
CDKN1A 6p21.2 Amplification 3/4
ECHS1 10q26.3 Deletion 4/6
MGMT 10q26.3 Deletion 5/6
TIMP2 17q25.3 Amplification 5/6
BDH 3q Amplification 6/7
FLJ20265 4p Amplification 6/7
PAO 10q Amplification 7/7
OPR1 20q Amplification 5/7
Table 3.
 
Clinicopathologic Features of Primary CoMs and Their Correlation with Amplification of CDKN1A and RUNX2
Table 3.
 
Clinicopathologic Features of Primary CoMs and Their Correlation with Amplification of CDKN1A and RUNX2
P027 Assay (n = 21)
With CDKN1A Amplification (n = 11) Without CDKN1A Amplification (n = 10) P With RUNX2 Amplification (n = 14) Without RUNX2 Amplification (n = 7) P
Sex 0.590* 0.562*
    Male 5 5 7 3
    Female 6 5 7 4
Age, y 0.654† 0.799†
    Median 66 56 59 55.5
    Range 20–95 40–76 20–95 40–74
Histology 0.857‡ 0.837‡
    Spindle 4 2 5 1
    Mixed 4 5 5 4
    Epithelioid 3 3 4 2
Survival at end of study period 0.356§ 0.329§
    Alive, n 10 5 11 4
    Dead, n 1 2 3 0
    Unknown, n 0 3 0 3
    Median, mo 36 36 36 48
    Range, mo 12–108 2.5–72 2.5–108 12–72
Table st1, XLS
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×