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Cancer Research 67, 2632, March 15, 2007. doi: 10.1158/0008-5472.CAN-06-4152
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Genome-Wide Loss of Heterozygosity and Copy Number Analysis in Melanoma Using High-Density Single-Nucleotide Polymorphism Arrays

Mitchell Stark and Nicholas Hayward

Oncogenomics Laboratory, Queensland Institute of Medical Research, Herston, Queensland, Australia

Requests for reprints: Nicholas Hayward, Oncogenomics Laboratory, Queensland Institute of Medical Research, 300 Herston Road, Herston, Brisbane, QLD 4006, Australia. Phone: 61-7-33620306; Fax: 61-7-38453508; E-mail: Nick.Hayward{at}qimr.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although a number of genes related to melanoma development have been identified through candidate gene screening approaches, few studies have attempted to conduct such analyses on a genome-wide scale. Here we use Illumina 317K whole-genome single-nucleotide polymorphism arrays to define a comprehensive allelotype of melanoma based on loss of heterozygosity (LOH) and copy number changes in a panel of 76 melanoma cell lines. In keeping with previous reports, we found frequent LOH on chromosome arms 9p (72%), 10p (55%), 10q (55%), 9q (49%), 6q (43%), 11q (43%), and 17p (41%). Tumor suppressor genes (TSGs) can be identified through homozygous deletion (HD). We detected 174 HDs, the most common of which targeted CDKN2A (n = 33). The second highest frequency of HD occurred in PTEN (n = 8), another well known melanoma TSG. HDs were also common for PTPRD (n = 7) and HDAC4 (n = 3), TSGs recently found to be mutated or deleted in other cancer types. Analysis of other HDs and regions of LOH that we have identified might lead to the characterization of further melanoma TSGs. We noted 197 regional amplifications, including some centered on the melanoma oncogenes MITF (n = 9), NRAS (n = 3), BRAF (n = 3), and CCND1 (n = 3). Other amplifications potentially target novel oncogenes important in the development of a subset of melanomas. The numerous focal amplifications and HDs we have documented here are the first step toward identifying a comprehensive catalog of genes involved in melanoma development, some of which may be useful prognostic markers or targets for therapies to treat this disease. [Cancer Res 2007;67(6):2632–42]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several genes have been shown to be mutated in melanoma, mostly through candidate gene screening approaches (reviewed in ref. 1). Elucidation of such genes has important implications for defining melanoma subtypes (2) and for tailoring treatment (e.g., MEK, KIT, or BRAF inhibitors).

Although many loss of heterozygosity (LOH) studies have been conducted in melanoma, most have focused on small chromosomal regions or a limited number of chromosomes. Only one study (3) has attempted to carry out a genome-wide allelotype, using one to three microsatellite markers from each chromosome arm. Conventional chromosome-based comparative genomic hybridization (CGH) has been used to study melanomas of different subtypes (4–7), and a limited number of studies have looked at array-based CGH (aCGH) in murine (8), swine (9), and human (2, 10) melanomas. The latter studies have led to the identification of CDK4, CCND1, and KIT amplifications in a subset of malignant melanoma. Although aCGH is adequate for detecting high level amplifications and homozygous deletions (HD), it grossly underestimates the level of LOH (11). In contrast, the use of high-density single-nucleotide polymorphism (SNP) arrays has proved to be a superior approach to defining genome-wide LOH and copy number changes in a wide range of tumor types (e.g., refs. 12–20).

Only one study to date has assessed LOH and copy number changes in melanoma using SNP arrays (21). Eight melanoma cell lines from the NCI60 series of tumor lines were analyzed using Affymetrix 100K SNP chips (Affymetrix, Santa Clara, CA). Although these data are publicly available,1 the authors did not present a full summary of the results but rather focused on the key finding of MITF amplification in some samples.

Here, we use high-density whole-genome SNP arrays, with an average inter-SNP spacing of 9 kb to define a comprehensive allelotype of melanoma based on LOH and copy number changes. Tumor suppressor genes (TSG) can be identified through HD. We detected 174 HDs in a panel of 76 melanoma cell lines, the most common of which centered on CDKN2A, PTEN, PTPRD, and HDAC4. The latter two loci have not previously been shown to play a role in melanoma development. Other HDs are likely to point to the location of additional uncharacterized melanoma TSGs. Oncogenes can be identified by amplification. We detected 197 focal amplifications targeting between 1 and 131 genes in each amplicon. Among these were increased copy numbers of BRAF, CCND1, MDM2, MITF, NRAS, and PIK3CA. Other amplifications potentially target novel melanoma oncogenes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and DNA extraction. The 76 melanoma cell lines used in this study were derived from primary cutaneous melanomas or melanoma metastases, as described previously (22). DNA was extracted using QIAGEN QIamp Blood Maxi kits according to the manufacturer's instructions (Qiagen, Hilden, Germany).

Mutation analysis of cell lines. Mutations in the BRAF, NRAS, HRAS, KRAS, CDKN2A, and PTEN genes have previously been described in this panel of cell lines (22–24).

SNP analysis. The Infinium II assay was done using Illumina Sentrix HumanHap300 genotyping BeadChip arrays (317K, TagSNP Phase I, v1.1) according to the manufacturer's specifications (Illumina, San Diego, CA). Briefly, 750 ng of genomic DNA were amplified at 37°C overnight, using solutions WG-AMM and WG-MP1. After overnight incubation, the amplified DNA was fragmented using WG-FRG and precipitated with isopropanol after the addition of WG-PA1. The dried precipitated pellet was then resuspended in WG-RA1 and hybridized to a beadchip along with WG-RA1 and formamide. The arrays were then incubated overnight at 48°C, after which they underwent single-base extension on a Teflow chamber rack system (Tecan, Männedorf, Switzerland) using WG-XC1, WG-XC2, and WG-TEM. After the single-base extension step, the beadchips were stained with WG-LTM and WG-ATM, dried for 1 h, then imaged using a BeadArray Reader (Illumina). Image data was analyzed using Beadstudio 2.0 (Illumina). All genomic positions were based upon hg17 from the UCSC Genome Browser.2 For additional details and example outputs, refer to Peiffer et al. (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TSG and oncogene mutation status. Table 1 summarizes the status of several key oncogenes and TSGs in the panel of 76 melanoma cell lines. At least 54 (71%) had CDKN2A defects, 48 (63%) had BRAF mutations, 10 (13%) had NRAS mutations (none had mutations in HRAS or KRAS), and 17 (22%) had PTEN defects. No cell line had a concomitant defect in NRAS and BRAF, or NRAS and PTEN. All cell lines with a PTEN defect also carried a BRAF mutation.


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  		Table 1. Summary of melanoma cell lines and mutation status of selected oncogenes and TSGs

 
We assessed the site or histotype of the primary melanomas in the patients from whom the cell lines were derived. For 19 of the cell lines, the site of the primary lesion was unknown; this included five patients who presented with metastatic disease and had no detectable primary tumor (i.e., occult primary). Based on the categorization of Curtin et al. (2), 54 (95%) of the remaining 57 lines were derived from nonchronic sun-damaged lesions, one was from a chronic sun-damaged site (the nose), one was an acral (plantar) melanoma, and one was from a buccal mucosa melanoma (Table 1). Thus, although the bulk of our data reflect nonchronic sun-damaged melanomas, the inclusion of isolated examples of melanomas of other histotypes allows some salient comparisons to be made. The cell line derived from an acral melanoma is wild-type for all of the above genes. The cell line derived from a mucosal melanoma carried a missense mutation affecting CDKN2A (P114L) and an NRAS mutation (Q61L).

Whole chromosome arm copy number aberrations. Combined LOH/CGH revealed a number of recurrent copy number changes affecting whole chromosome arms (Table 2 ). Most common among the losses were both arms of chromosomes 9 and 10, which occurred in ~40% to 50% of all samples. The majority of these losses were due to hemizygous deletions, but 30% to 40% were due to copy number neutral LOH or a combination of copy number neutral LOH and hemizygous deletion. The next most frequent were losses of 17q (30%) and 17p (25%). Approximately 75% of the latter were the result of copy number neutral LOH, which contrasted with the 40% of losses on the q arm that occurred through this mechanism. The only other chromosome arm exhibiting complete loss in >20% of samples was 5q (24%), half of which were due to hemizygous deletion (Table 2).


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  		Table 2. Numbers of cell lines with whole chromosome arm copy number gains and losses

 
The most common copy number gains occurred for chromosome arms 2p, 6p, 7p, 7q, 19q, 20p, 20q, and 22q. Each were present in 20% to 30% of the cell lines. Trisomy for these chromosome arms was the most common copy number deviation, although the occasional cell line was found to harbor up to eight copies (Table 2).

Regional LOH analysis. The whole-genome SNP data were further interrogated for regions of LOH that occurred across smaller intervals than a whole chromosome arm. Cross-referencing the Database of Genomic Variants3 was done to eliminate calling common structural polymorphisms as small regions of LOH. Table 3 lists the cytobands and nucleotide intervals showing the most frequent regional LOH on each chromosome arm. The commonest occurred at 9p21.3 (position 20317754–25227815) and targeted the CDKN2A locus (72%). The next most frequent (55%) were LOH of 10q23.2-q21.33 (position 87491745–87930977), targeting PTEN, and 10pter-p15.1 (position 103934–5767329), targeting an as yet unidentified TSG. Overall levels of LOH of >40% also occurred at 6q25.1-q25.3, 9q21.2-q21.33, 11q22.3, and 17p13.1.


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  		Table 3. Most common regional LOH intervals

 
Homozygous deletions. In the panel of 76 melanoma cell lines, we detected a total of 174 HDs. Of these, 52 HDs seemed to target a single locus, 87 (50%) targeted more than one gene, and 35 of the HDs did not encompass the coding region of an annotated gene (human genome build hg17). HDs ranged in size from 78 kb to 12.8 Mb. Table 4 lists the chromosomal regions showing HD in one or more cell lines and the genes that reside in these regions. The full list of all HDs, including those that did not affect annotated genes, is given in Supplementary Table S1. On average, each cell line carried 2.3 HDs (range, 0–11; Table 1). Notably, the mucosal and acral melanoma cell lines were among those with the lowest frequency of deletions, having zero and one HD, respectively. Sixteen of the HDs occurred in more than one cell line (Table 4). The most common HD (43%, 33 of 76 cell lines) affected CDKN2A (Fig. 1A ) and extended to variable numbers of neighboring genes on 9p22-p21, including CDKN2B, DMRTA1, ELAV2, the IFN cluster, KIAA1797, MTAP, MLLT3, PTPLAD2, and TUSC1 (Table 4). The next most common deletions affected the PTEN (Fig. 1B), PTPRD (Fig. 1C), and HDAC4 (Fig. 1D) loci and occurred in eight (11%), seven (9%), and three (4%) cell lines, respectively. With the exception of one intragenic microdeletion in PTEN we reported previously (24), there was complete correlation between previous CDKN2A and PTEN deletions detected by PCR sequencing (23, 24) and those detected here by aCGH.


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  		Table 4. Summary of HDs that include an annotated gene

 

Figure 1
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  		Figure 1. Examples of HDs detected using Illumina 317K BeadChip SNP arrays. In each case, log2 R ratios for the SNPs are plotted on the X axis above the chromosome ideogram. Most values are centered around 0, indicating diploid copy number. HDs appear as clusters of SNPs with highly negative log2 ratios, whereas amplifications are highlighted by an increase in log2 R. A, HD of CDKN2A (boxed). B, HD of PTEN (boxed; note a second HD near the centromere). C, HD of PTPRD (boxed; this cell line also has a CDKN2A HD). D, HD of HDAC4 (boxed; at the edge of a small region of hemizygosity).

 
Regional amplifications. There were a total of 197 focal (i.e., subchromosomal arm) amplifications in the panel of melanoma cell lines. These were generally much larger than the HDs and ranged in size from 228 kb to 28.8 Mb. The number of amplicons per cell line ranged from 0 to 29, with the most occurring in the mucosal melanoma (Table 1). Supplementary Table S2 lists the chromosomal regions showing focal amplification in one or more cell lines and the genes that reside in these regions. With a few exceptions, including MITF (Fig. 2A ), BRAF, CDH9, DKFZp564N2472, GBE1, LRFN5, NAALADL2, NFIB, PTPRD, SESN3, SLC26A7, SNTG1, TMEFF2, and WDR72, the amplicons contained multiple genes (range, 1–131). Several recurring amplifications seemed to target the same genes (Supplementary Tables S2 and S3). The most common of these centered on MITF and was seen in nine (12%) samples. Others included genes such as ANK1 (Fig. 2B). Rare instances of focal amplification, including the BRAF, CCND1, MDM2, NRAS, and PIK3CA genes, were also observed (Supplementary Table S3). Notably, none of these amplifications of documented melanoma oncogenes occurred in the acral or mucosal melanoma cell lines. Among a large number of focal amplifications in the acral melanoma were amplifications that included MAP3K8 (Fig. 2C) and SMO (Supplementary Table S2). The numerous amplifications seen in the mucosal melanoma included CCNB2 and TERT.


Figure 2
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  		Figure 2. Examples of focal amplifications (see legend to Fig. 1). A, amplification of MITF (boxed). B, focal amplification of ANK1 (boxed). C, high-level focal amplification centered on MAP3K8 (boxed) within a larger region of amplification (note numerous other amplified regions on this chromosome from the acral melanoma). D, amplification of NRAS (boxed; note the large region of hemizygous deletion in the middle of the q arm).

 
Although we detected high-level focal amplification of the NRAS gene (e.g., Fig. 2D) in two cell lines that carried NRAS point mutations (A07 and CJM), there was generally poor correlation between copy number increases of BRAF, HRAS, KRAS, and NRAS, and concomitant mutations in these oncogenes. There were no focal amplifications affecting CDK2, CDK4, CDK6, HRAS, KRAS, KIT, or MYC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we provide the first comprehensive whole-genome allelotype for melanoma. Whole chromosome arm LOH was most common on 9p, 9q, 10p, and 10q and occurred in ~40% to 50% of all samples (Table 2). Of these 30% to 40% were due to copy number neutral LOH, which would be missed by conventional metaphase spread or BAC aCGH. When focal LOH was also taken into account (Table 3), the same four chromosome arms showed the highest overall frequencies of LOH (49–72%) in keeping with previous reports (e.g., refs. 3, 26–34). Overall LOH was next most common (>40%) on 6q, 11q, and 17p, once again, in support of prior studies (e.g., refs. 3, 30, 31, 34–36). Additionally, we observed 33% LOH on 5q, a chromosome not previously associated with harboring a putative TSG for melanoma.

The SNP arrays were very effective at detecting HDs. Exemplifying the power of this approach, we "rediscovered" the high frequency of HDs of CDKN2A and PTEN in melanoma (Fig. 1A and B). These genes were also the targets of the most recurrent regions of focal LOH (Table 3). We have also identified many regions of HD that putatively target other TSGs. In keeping with the analysis of other cancer genomes (37), we have also found that some HDs target fragile sites. We observed HDs in both the FHIT (FRA3B) and WWOX (FRA16D) loci in two cell lines (Table 4). Some of the genes in HDs that we have identified here in melanoma have recently been shown to be mutated in breast and colon cancers (38), for example, HDAC4 and C14ORF155, both listed as a candidate cancer gene for breast cancer, and PTPRD and GUCY1A2, both listed as candidate cancer gene for colon cancer. This suggests that these genes may also be the targets of the deletions in melanoma and imply that the genes might be general TSGs affecting tumorigenesis in a wide variety of cancer types. Similarly, we found other loci deleted in the melanoma cell line panel that belong to a number of gene families with members mutated in breast and colon cancers (38), for example, CD274, CDH18, CNTN5, DDX4, GRIN3A, KCNV2, LGR4, LRRN6C, MAGEC1, MAGEC2, MAGEC3, PCD11X, PCDH15, PLEKHA5, PRPF39, RFX3, SEMA6D, SLC1A1, and ZFP91. This supports a role for these gene families as general tumor suppressors.

Numerical chromosome copy number increases, and focal amplifications putatively target oncogenes. We found the most common (≥25%) whole chromosome arm copy number gains occurred for 7p, 20q, and 22q (Table 2). This concurs with the conventional chromosome CGH data for these chromosome arms in various melanoma subtypes (4–7, 39). Regional amplifications of KIT, MITF, BRAF, NRAS, HRAS, KRAS, MDM2, CDK4, CCND1, MYC, and PIK3CA have been reported in some melanomas. Here we found amplification of MITF in nine cell lines (12%) in keeping with the frequency of 10% in primary melanomas and 15% to 20% in melanoma metastases reported previously (21). KIT amplifications and/or mutations have been found in 28% to 39% of acral, mucosal, and chronic sun-damaged melanomas but occur very rarely in nonchronic sun-damaged melanomas (10, 40, 41). In support of the latter finding, we saw no amplifications of KIT in our panel of predominantly nonchronic sun-damaged cell lines. CDK4 amplifications were found in 11 (9%) samples (six acral, four mucosal, and one chronic sun-damaged melanomas) in a study of 126 melanomas of various histologic types (2). In another report, CDK4 amplification was documented in 3 (6%) of 51 melanoma metastases (42). In two of these tumors, the amplicon was bipartite, with the second amplification peak centered on the MDM2 gene. None of the three samples with amplification of either gene had defects in CDKN2A (affecting either p16 or p14), indicating that amplification of the CDK4 and MDM2 genes is an alternative way for tumor cells to simultaneously inactivate the pRb and p53 pathways. We found a regional amplification of MDM2 (that did not extend to encompass CDK4) in a single cell line, MM595, in which the entire CDKN2A locus is homozygously deleted.

CCND1 is also a commonly amplified locus in melanoma, occurring most frequently in the acral subtype (2). This locus was amplified in three of our melanoma cell lines (A06, MM548, and SKMEL5). PIK3CA mutations have been found in 1 of 118 primary melanomas and 1 of 34 melanoma cell lines analyzed (43), as well as in secondary melanomas from 2 of 87 patients tested (44). Only one of the melanoma cell lines studied here carried a PIK3CA mutation (D17; ref. 43), but because PIK3CA activation can also occur through gene amplification (45), we assessed whether regional amplifications of this locus were evident from the aCGH data. One cell line was observed to have PIK3CA amplification (MM636).

Others have reported increased copies of MYC, on chromosome arm 8q, in 15 (37%) of a series of 41 primary or secondary melanomas analyzed by fluorescence in situ hybridization (46). In contrast, we did not observe focal amplifications of MYC in any cell line, and only 14% of samples had increased copies of 8q (Table 2). No regional amplifications of the above-mentioned genes were seen in either the acral or mucosal cell line in our panel. However, both of these lines carried a large number of amplifications at other chromosomal sites. Indeed, the mucosal melanoma possessed the largest number of focal amplifications (n = 29), supporting previous observations (2).

The large number of amplifications and HDs we have documented here provides a platform for further studies aimed at identifying novel melanoma oncogenes and TSGs and understanding how they contribute to melanoma development. Moreover, some of these may prove to be useful clinically, either as prognostic markers or as targets for therapies to treat melanoma.


   Acknowledgments
 
Grant support: National Health and Medical Research Council of Australia (NHMRC), Queensland Cancer Fund, and Senior Principal Research Fellowship of NHMRC (N. Hayward).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Jane Palmer, Sandra Pavey, and Cathy Lanagan for tracking clinical details of the patients and the groups of Peter Parsons and Chris Schmidt for their efforts in establishing the majority of melanoma cell lines used in this study.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

1 http://www.ncbi.nlm.nih.gov/geo (accession number GSE2520) Back

2 http://genome.ucsc.edu/ Back

3 http://projects.tcag.ca/variation/ Back

Received 11/ 9/06. Revised 1/ 4/07. Accepted 1/ 9/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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