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]

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 (47), 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. 1220).

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

1

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

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.

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 (2224).

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).

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.

Table 1.

Summary of melanoma cell lines and mutation status of selected oncogenes and TSGs

Cell linesSite primary melanomaNo. HDsNo. ampliconsCDKN2APTENBRAFNRAS
A02 non-CSD wt wt V599E wt 
A04 non-CSD HD wt wt wt 
A06 non-CSD 11 HD HD V599E wt 
A07 non-CSD E2K wt wt G12S 
A12 non-CSD     
A13 non-CSD W110Stop HD V599E wt 
A15 non-CSD wt (methylated) wt V599K wt 
AF6  splice mutation splice mutation V599E wt 
C-32 non-CSD HD HD V599E wt 
CJM  HD wt wt Q61K 
D01 occult HD wt wt Q61K 
D03 non-CSD     
D04 non-CSD HD wt wt Q61L 
D05 non-CSD frameshift mutation wt V599E wt 
D08 non-CSD HD wt wt Q61L 
D10 acral wt wt wt wt 
D11 mucosal 29 P114L wt wt Q61L 
D14 non-CSD P114L HD V599E wt 
D17 non-CSD wt (methylated) wt V599E wt 
D20 non-CSD 16 wt HD V599E wt 
D22 non-CSD HD wt wt wt 
D24  HD wt wt wt 
D25 non-CSD wt wt V599E wt 
D28 occult P114L wt V599K wt 
D29  HD wt V599E wt 
D30 non-CSD     
D32  HD frameshift mutation V599E wt 
D35  11 splice mutation wt wt wt 
D36 non-CSD R80Stop HD V599E wt 
D38 occult wt (methylated) wt wt wt 
D40 non-CSD HD wt V599E wt 
D41 non-CSD wt wt V599E wt 
D49 non-CSD     
D54 non-CSD     
D64 non-CSD     
HT144  HD HD V599E wt 
JA  wt wt V599E wt 
MM96 non-CSD wt (methylated) wt V599E wt 
MM127 non-CSD HD wt wt G13R 
MM138 non-CSD HD    
MM200 non-CSD wt F56I V599E wt 
MM229 non-CSD HD wt L596S wt 
MM253 non-CSD HD wt V599E wt 
MM266 non-CSD     
MM329 non-CSD 10 wt wt wt wt 
MM369 non-CSD R80Stop wt V599E wt 
MM370 non-CSD HD wt V599E wt 
MM384 occult P48L wt V599E wt 
MM386 non-CSD wt (methylated) HD V599E wt 
MM396 non-CSD P81L wt V599E wt 
MM409 non-CSD HD wt V599E wt 
MM415 non-CSD wt wt wt Q61L 
MM418 non-CSD HD wt V599E wt 
MM426 non-CSD E88stop wt V599E wt 
MM466 non-CSD HD wt V599E wt 
MM472 CSD HD P30L V599E wt 
MM473 non-CSD H83Y wt V599E wt 
MM485 non-CSD W110Stop wt wt Q61R 
MM488 non-CSD HD HD V599E wt 
MM537 non-CSD HD wt V599E wt 
MM540 non-CSD wt (methylated) wt V599E wt 
MM548 non-CSD delR99 wt V599E wt 
MM595 non-CSD HD wt V599E wt 
MM603  wt wt V599E wt 
MM604 non-CSD 11 wt HD V599E wt 
MM622 non-CSD frameshift mutation L139Stop V599E wt 
MM628 occult P114L wt wt G13R 
MM636 non-CSD     
MM647 non-CSD aberrant transcript wt wt wt 
MM648 non-CSD 10 HD wt V599E wt 
MM649 non-CSD wt (methylated) wt V599E wt 
MW  HD wt V599R wt 
RPMI7932      
SKMEL28  wt T167A V599E wt 
SKMEL5  HD del exon 1 V599E wt 
WW  HD wt V599E wt 
Cell linesSite primary melanomaNo. HDsNo. ampliconsCDKN2APTENBRAFNRAS
A02 non-CSD wt wt V599E wt 
A04 non-CSD HD wt wt wt 
A06 non-CSD 11 HD HD V599E wt 
A07 non-CSD E2K wt wt G12S 
A12 non-CSD     
A13 non-CSD W110Stop HD V599E wt 
A15 non-CSD wt (methylated) wt V599K wt 
AF6  splice mutation splice mutation V599E wt 
C-32 non-CSD HD HD V599E wt 
CJM  HD wt wt Q61K 
D01 occult HD wt wt Q61K 
D03 non-CSD     
D04 non-CSD HD wt wt Q61L 
D05 non-CSD frameshift mutation wt V599E wt 
D08 non-CSD HD wt wt Q61L 
D10 acral wt wt wt wt 
D11 mucosal 29 P114L wt wt Q61L 
D14 non-CSD P114L HD V599E wt 
D17 non-CSD wt (methylated) wt V599E wt 
D20 non-CSD 16 wt HD V599E wt 
D22 non-CSD HD wt wt wt 
D24  HD wt wt wt 
D25 non-CSD wt wt V599E wt 
D28 occult P114L wt V599K wt 
D29  HD wt V599E wt 
D30 non-CSD     
D32  HD frameshift mutation V599E wt 
D35  11 splice mutation wt wt wt 
D36 non-CSD R80Stop HD V599E wt 
D38 occult wt (methylated) wt wt wt 
D40 non-CSD HD wt V599E wt 
D41 non-CSD wt wt V599E wt 
D49 non-CSD     
D54 non-CSD     
D64 non-CSD     
HT144  HD HD V599E wt 
JA  wt wt V599E wt 
MM96 non-CSD wt (methylated) wt V599E wt 
MM127 non-CSD HD wt wt G13R 
MM138 non-CSD HD    
MM200 non-CSD wt F56I V599E wt 
MM229 non-CSD HD wt L596S wt 
MM253 non-CSD HD wt V599E wt 
MM266 non-CSD     
MM329 non-CSD 10 wt wt wt wt 
MM369 non-CSD R80Stop wt V599E wt 
MM370 non-CSD HD wt V599E wt 
MM384 occult P48L wt V599E wt 
MM386 non-CSD wt (methylated) HD V599E wt 
MM396 non-CSD P81L wt V599E wt 
MM409 non-CSD HD wt V599E wt 
MM415 non-CSD wt wt wt Q61L 
MM418 non-CSD HD wt V599E wt 
MM426 non-CSD E88stop wt V599E wt 
MM466 non-CSD HD wt V599E wt 
MM472 CSD HD P30L V599E wt 
MM473 non-CSD H83Y wt V599E wt 
MM485 non-CSD W110Stop wt wt Q61R 
MM488 non-CSD HD HD V599E wt 
MM537 non-CSD HD wt V599E wt 
MM540 non-CSD wt (methylated) wt V599E wt 
MM548 non-CSD delR99 wt V599E wt 
MM595 non-CSD HD wt V599E wt 
MM603  wt wt V599E wt 
MM604 non-CSD 11 wt HD V599E wt 
MM622 non-CSD frameshift mutation L139Stop V599E wt 
MM628 occult P114L wt wt G13R 
MM636 non-CSD     
MM647 non-CSD aberrant transcript wt wt wt 
MM648 non-CSD 10 HD wt V599E wt 
MM649 non-CSD wt (methylated) wt V599E wt 
MW  HD wt V599R wt 
RPMI7932      
SKMEL28  wt T167A V599E wt 
SKMEL5  HD del exon 1 V599E wt 
WW  HD wt V599E wt 

NOTE: Blanks indicate missing information. Abbreviations: CSD, chronic sun-damaged; wt, wild-type.

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).

Table 2.

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

Chromosome armComplete loss
Partial loss*
Gains
HemiNeutMixedTotalHemiNeutMixedTotal345678Total
1p 7 (9) 11 (14)     4 (5) 
1q 6 (8) 11 13 (17) 10    13 (17) 
2p 7 (9) 16 17 (22)   15 (20) 
2q 7 (9) 13 14 (18)    7 (9) 
3p 3 (4) 11 13 (17)    5 (7) 
3q 2 (3) 14 18 (24)     4 (5) 
4p 7 (9) 19 24 (32)    4 (5) 
4q 6 (8) 15 19 (25)       0 (0) 
5p 10 (13) 19 20 (26)     8 (11) 
5q 18 (24) 14 15 (20)     2 (3) 
6p 5 (6) 18 18 (24) 11    15 (20) 
6q 9 (12) 10 13 (17)       0 (0) 
7p 5 (6) 7 (9) 15    20 (26) 
7q 5 (6) 4 (5) 12   18 (24) 
8p 10 (13) 19 20 (26)    4 (5) 
8q 2 (3) 16 17 (22)   11 (14) 
9p 20 10 34 (45) 8 (11)       0 (0) 
9q 19 11 33 (43) 8 (11)      1 (1) 
10p 24 36 (47) 6 (8)       1 (1) 
10q 27 39 (51) 7 (9)       0 (0) 
11p 12 (16) 24 26 (34)       0 (0) 
11q 10 (13) 15 17 (22)       0 (0) 
12p 3 (4) 14 22 (29)      8 (11) 
12q 6 (8) 13 21 (28)      5 (7) 
13q 10 (13) 13 (17)      4 (5) 
14q 9 (12) 10 19 (25)       0 (0) 
15q 5 (6) 11 (14)     6 (8) 
16p 2 (3) 18 19 (25)    8 (11) 
16q 8 (11) 12 14 (18)     5 (7) 
17p 14 19 (25) 3 (4) 12     13 (17) 
17q 14 23 (30) 3 (4)      7 (9) 
18p 13 (17) 13 20 (26)      1 (1) 
18q 1 (1) 12 24 (32)       0 (0) 
19p 6 (8) 4 (5) 10     11 (14) 
19q 4 (5) 5 (7) 12    15 (20) 
20p 5 (6) 5 (7) 12    15 (20) 
20q 1 (1) 0 (0) 14   19 (25) 
21q 4 (5) 18 20 (26)     3 (4) 
22q 5 (6) 1 (1) 20     22 (29) 
Chromosome armComplete loss
Partial loss*
Gains
HemiNeutMixedTotalHemiNeutMixedTotal345678Total
1p 7 (9) 11 (14)     4 (5) 
1q 6 (8) 11 13 (17) 10    13 (17) 
2p 7 (9) 16 17 (22)   15 (20) 
2q 7 (9) 13 14 (18)    7 (9) 
3p 3 (4) 11 13 (17)    5 (7) 
3q 2 (3) 14 18 (24)     4 (5) 
4p 7 (9) 19 24 (32)    4 (5) 
4q 6 (8) 15 19 (25)       0 (0) 
5p 10 (13) 19 20 (26)     8 (11) 
5q 18 (24) 14 15 (20)     2 (3) 
6p 5 (6) 18 18 (24) 11    15 (20) 
6q 9 (12) 10 13 (17)       0 (0) 
7p 5 (6) 7 (9) 15    20 (26) 
7q 5 (6) 4 (5) 12   18 (24) 
8p 10 (13) 19 20 (26)    4 (5) 
8q 2 (3) 16 17 (22)   11 (14) 
9p 20 10 34 (45) 8 (11)       0 (0) 
9q 19 11 33 (43) 8 (11)      1 (1) 
10p 24 36 (47) 6 (8)       1 (1) 
10q 27 39 (51) 7 (9)       0 (0) 
11p 12 (16) 24 26 (34)       0 (0) 
11q 10 (13) 15 17 (22)       0 (0) 
12p 3 (4) 14 22 (29)      8 (11) 
12q 6 (8) 13 21 (28)      5 (7) 
13q 10 (13) 13 (17)      4 (5) 
14q 9 (12) 10 19 (25)       0 (0) 
15q 5 (6) 11 (14)     6 (8) 
16p 2 (3) 18 19 (25)    8 (11) 
16q 8 (11) 12 14 (18)     5 (7) 
17p 14 19 (25) 3 (4) 12     13 (17) 
17q 14 23 (30) 3 (4)      7 (9) 
18p 13 (17) 13 20 (26)      1 (1) 
18q 1 (1) 12 24 (32)       0 (0) 
19p 6 (8) 4 (5) 10     11 (14) 
19q 4 (5) 5 (7) 12    15 (20) 
20p 5 (6) 5 (7) 12    15 (20) 
20q 1 (1) 0 (0) 14   19 (25) 
21q 4 (5) 18 20 (26)     3 (4) 
22q 5 (6) 1 (1) 20     22 (29) 

NOTE: Percentages of totals are given in parentheses.

Abbreviations: Hemi, hemizygous deletion; Neut, copy number neutral LOH; Mixed, mixture of copy number neutral LOH and hemizygous loss.

*

Partial loss occurring in a proportion of cells (i.e., mosaicism).

Only uniform copy number gains across the length of the chromosome arm have been included, thus, for example, an arm that was partly four copies and partly five copies would not appear in the table.

Number of chromosome arms.

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.

Table 3.

Most common regional LOH intervals

Chromosome armSRO within cytoband(s)Start base positionEnd base positionStart base positionEnd base positionnTotal (%)
1p 31.1 81688407 8445590   13 20 (26) 
1q 42.2 228366540 228914590   12 (16) 
2p 25.2 and 24.3 5805898 6119949 15755054 16984942 12 (16) 
2q 37.3 239293533 242759899   12 19 (25) 
3p 21.31 45950987 49696536   14 17 (22) 
3q 26.31 176577059 176998125   10 12 (16) 
4p 15.1 32418953 33463430   15 (20) 
4q 32.1 and 32.3 157984245 158621187 164832073 166472504 10 16 (21) 
5p 14.3 22699011 23194868   16 (21) 
5q 11.2 58447527 58681562   25 (33) 
6p 22.2-21.33 29361735 32298598   8 (11) 
6q 25.1-25.3 152362786 155321679   24 33 (43) 
7p 21.3-21.1 7187594 17555849   8 (11) 
7q 22.3-31.1 105769871 111045610   13 (17) 
8p 12 31614916 32931772   15 (20) 
8q 11.21-11.22 and 21.3 48136481 51946973 89271300 90686126 5 (7) 
9p 21.3 20317754 25227815   21 55 (72) 
9q 21.2-21.33 77651737 83134614   37 (49) 
10p ter-15.1 103934 5767329   42 (55) 
10q 23.2-23.31 87491745 87930977   42 (55) 
11p 14.1 and 11.2 29001470 29431689 45266986 46642836 21 (28) 
11q 22.3 102626683 105738304   23 33 (43) 
12p 12.1 20781209 21633573   10 (13) 
12q 21.32 and 24.31 87076009 87733383 119303242 123506991 10 16 (21) 
13q 33.3-34 108060380 114108121   17 (22) 
14q 32.2-ter 98443619 106345097   15 (20) 
15q 21.1 46980083 47525525   11 16 (21) 
16p 12.1 23645856 24292120   7 (9) 
16q 24.3 87580066 88668978   14 22 (29) 
17p 13.1 7167436 8239718   12 31 (41) 
17q 21.32 and 22 42660346 44186025 48473172 50089530 30 (39) 
18p 11.32 59836 1265507   15 (20) 
18q 22.1 and 22.3 62097585 65010233 67876215 68301919 7 (9) 
19p 13.3 217034 1822341   12 (16) 
19q 13.32 52070865 53777522   10 (13) 
20p 13 and 12.3 1110370 3654863 6717533 8499344 12 (16) 
20q 13.2 52853092 53531064   4 (5) 
21q 21.1 19744931 21665674   13 17 (22) 
22q 12.2 29897517 30554739   12 (16) 
Chromosome armSRO within cytoband(s)Start base positionEnd base positionStart base positionEnd base positionnTotal (%)
1p 31.1 81688407 8445590   13 20 (26) 
1q 42.2 228366540 228914590   12 (16) 
2p 25.2 and 24.3 5805898 6119949 15755054 16984942 12 (16) 
2q 37.3 239293533 242759899   12 19 (25) 
3p 21.31 45950987 49696536   14 17 (22) 
3q 26.31 176577059 176998125   10 12 (16) 
4p 15.1 32418953 33463430   15 (20) 
4q 32.1 and 32.3 157984245 158621187 164832073 166472504 10 16 (21) 
5p 14.3 22699011 23194868   16 (21) 
5q 11.2 58447527 58681562   25 (33) 
6p 22.2-21.33 29361735 32298598   8 (11) 
6q 25.1-25.3 152362786 155321679   24 33 (43) 
7p 21.3-21.1 7187594 17555849   8 (11) 
7q 22.3-31.1 105769871 111045610   13 (17) 
8p 12 31614916 32931772   15 (20) 
8q 11.21-11.22 and 21.3 48136481 51946973 89271300 90686126 5 (7) 
9p 21.3 20317754 25227815   21 55 (72) 
9q 21.2-21.33 77651737 83134614   37 (49) 
10p ter-15.1 103934 5767329   42 (55) 
10q 23.2-23.31 87491745 87930977   42 (55) 
11p 14.1 and 11.2 29001470 29431689 45266986 46642836 21 (28) 
11q 22.3 102626683 105738304   23 33 (43) 
12p 12.1 20781209 21633573   10 (13) 
12q 21.32 and 24.31 87076009 87733383 119303242 123506991 10 16 (21) 
13q 33.3-34 108060380 114108121   17 (22) 
14q 32.2-ter 98443619 106345097   15 (20) 
15q 21.1 46980083 47525525   11 16 (21) 
16p 12.1 23645856 24292120   7 (9) 
16q 24.3 87580066 88668978   14 22 (29) 
17p 13.1 7167436 8239718   12 31 (41) 
17q 21.32 and 22 42660346 44186025 48473172 50089530 30 (39) 
18p 11.32 59836 1265507   15 (20) 
18q 22.1 and 22.3 62097585 65010233 67876215 68301919 7 (9) 
19p 13.3 217034 1822341   12 (16) 
19q 13.32 52070865 53777522   10 (13) 
20p 13 and 12.3 1110370 3654863 6717533 8499344 12 (16) 
20q 13.2 52853092 53531064   4 (5) 
21q 21.1 19744931 21665674   13 17 (22) 
22q 12.2 29897517 30554739   12 (16) 

NOTE: Total refers to combined number of samples with regional LOH and whole chromosome arm LOH (Table 2).

Abbreviations: SRO, smallest region of overlapping LOH; n, number of samples showing the particular regional LOH.

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.

Table 4.

Summary of HDs that include an annotated gene

Cell lineCytobandStart ntEnd ntSize (nt)Genes within HD
D35 1p35.2 31140795 31691124 550330 PUM1 FAM77C WDR57 ZCCHC17 FABP3 SERINC2 
WW 1p31.2 66531644 66921109 389466 PDE4B SGIP1     
MM548 1p21.3 97732018 98107902 375885 DPYD      
D28 1q31.3 193441431 193628794 187364 CFH CFHR3 CFHR1 CFHR4   
D01 1q44 244079149 244245509 166361 OR2G3 OR13G1 OR6F1 OR5AY1   
C-32 2p25.1 7064772 7441255 376484 RNF144      
MM386 2q32.1 188169003 188683293 514291 TFPI      
D04 2q33.3-q34 212189700 212941892 752193 ERBB4      
MM603 2q37.3 239601090 239845248 244159 HDAC4      
MM426 2q37.3 239716794 240039566 322773 HDAC4      
MM473 2q37.3 239716794 240111974 395181 HDAC4      
MM370 3p14.2 60180460 60857999 677540 FHIT      
MM253 3p14.2 60200880 60697129 496250 FHIT      
D22 3q13.31 117290277 117667176 376900 LSAMP      
D35 3q25.1 152622710 153335910 713201 MED12L IGSF10 AADACL2 AADAC SUCNR1  
D10 4p16.3 63508 141601 78094 ZNF595 ZNF718     
A13 4q22 93683540 94526401 842862 GRID2      
D14 4q26 117898191 118775563 877373 TRAM1L1      
A06 5p15.1-p14.3 17916022 19862376 1946355 CDH18      
D20 5q11.1 49945313 50116451 171139 PARP8      
D64 5q11.2 55069845 55450713 380869 DDX4 IL31RA IL6ST IL6ST ANKRD55  
D64 5q11.2 56163623 56702257 538635 MGC33648 MIER3 GPBP1    
WW 6p24.1 12496151 13168554 672404 PHACTR1      
A04 6q25.3 155816691 157219651 1402961 NOX3 ARID1B     
D35 6q26 162324738 162459269 134532 PARK2      
D29 6q27 167775932 168154636 378705 LOC401286 C6orf123 MLLT4    
MM370 7q21.11 77758447 78217314 458868 MAGI2      
D29 8q11.21 51697600 52226926 529327 SNTG1      
MM384 8q12.3 64209391 65473754 1264364 YTHDF3      
D54 9p24.3 194201 1248905 1054705 C9orf66 DOCK8 ANKRD15 DMRT1 DMRT3 DMRT2 
RPMI7932 9p24.3 1463302 14264056 12800755 SMARCA2 to NFIB     
MM253 9p24.1 5170982 5520017 349036 INSL6 to PDCD1LG2    
D54 9p24.1-p23 8021935 11421917 3399983 PTPRD      
MM253 9p24.1-p23 8892753 10148149 1255397 PTPRD      
A06 9p23 9159552 9438088 278537 PTPRD      
CJM 9p23 9297216 9630335 333120 PTPRD      
D14 9p23 9565540 9910033 344494 PTPRD      
D35 9p23 10422363 13999610 3577248 PTPRD TYRP1 C9orf150 MPDZ   
D24 9p23 12892159 13169615 277457    MPDZ   
A02 9p22.3-p22.2 15750988 16629077 878090 C9orf93 BNC2     
A15 9p22.3-p22.2 16296523 16772915 476393  BNC2     
MM540 9p22.3-p22.2 16296523 16903197 606675  BNC2 LOC648570    
D35 9p22.3-p22.2 16347674 17196306 848633 C9orf39 BNC2 LOC648570    
D32 9p22.3-p22.2 16443145 17390701 947557 C9orf39 BNC2 LOC648570    
D01 9p22.1-p21.3 19791657 22197037 2405381 MLLT3 to CDKN2B    
D35 9p21.3 20316948 25319508 5002561 MLLT3 to   ELAVL2  
D54 9p21.3 20857809 21189680 331872 KIAA1797 to IFNA4     
D24 9p21.3 20903989 23401162 2497174 KIAA1797 to  DMRTA1   
MM537 9p21.3 21119246 22774110 1654865 IFNW1 to  DMRTA1   
MW 9p21.3 21119246 23297724 2178479 IFNW1 to  DMRTA1   
D29 9p21.3 21154463 25319508 4165046 IFNA21 to   ELAVL2  
CJM 9p21.3 21397250 22197037 799788 IFNA8 to CDKN2B    
HT144 9p21.3 21507302 22128823 621522 MTAP CDKN2A CDKN2B    
MM138 9p21.3-p21.2 21523610 26323942 4800333 MTAP CDKN2A CDKN2B DMRTA1 ELAVL2 TUSC1 
MM127 9p21.3 21572720 22064580 491861 MTAP CDKN2A CDKN2B    
MM418 9p21.3 21572720 22064580 491861 MTAP CDKN2A CDKN2B    
HT144 9p21.3 21572720 22182093 609374 MTAP CDKN2A CDKN2B    
D04 9p21.3 21578603 22631633 1053031 MTAP CDKN2A CDKN2B DMRTA1   
MM472 9p21.3 21673289 22280617 607329 MTAP CDKN2A CDKN2B    
D32 9p21.3 21673289 23032805 1359517 MTAP CDKN2A CDKN2B DMRTA1   
MM229 9p21.3 21690220 22064569 374350 MTAP CDKN2A CDKN2B    
MM253 9p21.3 21779250 22131886 352637 MTAP CDKN2A CDKN2B    
MM540 9p21.3 21805166 21998659 193494 MTAP CDKN2A CDKN2B    
WW 9p21.3 21805166 21998659 193494 MTAP CDKN2A CDKN2B    
A04 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
C-32 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
D40 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
HT144 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
MM418 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
A06 9p21.3 21805166 22326954 521789 MTAP CDKN2A CDKN2B    
D08 9p21.3 21805166 22326954 521789 MTAP CDKN2A CDKN2B    
MM266 9p21.3 21805166 23243097 1437932 MTAP CDKN2A CDKN2B    
MM466 9p21.3 21805166 23432605 1627440 MTAP CDKN2A CDKN2B DMRTA1   
D49 9p21.3 21857756 22197037 339282  CDKN2A CDKN2B    
D54 9p21.3 21857756 22197037 339282  CDKN2A CDKN2B    
MM370 9p21.3 21857756 22197037 339282  CDKN2A CDKN2B    
MM648 9p21.3 21998659 22280617 281959 MTAP CDKN2A CDKN2B DMRTA1   
D22 9p21.3 21998659 23813162 1814504  CDKN2A CDKN2B DMRTA1 ELAVL2  
MM266 9p21.3 23401162 23864047 462886     ELAVL2  
WW 9p21.1 28439733 28902923 463191 LRRN6C      
WW 9p13.2 37869040 38062699 193660 MCART1 SHB     
A06 9q21.13 74224436 74417045 192610 RORB      
A13 9q31.1 101353456 101682714 329259 RNF20 GRIN3A PPP3R2    
D40 9q33.1 116760456 117221359 460904 ASTN2      
D32 9q33.2-q33.3 122191507 123462916 1271410 PTGS1 to DENND1A    
MM138 9q33.2 122382671 122817077 434407 OR1N2 to RABGAP1    
D05 10p15.3 103934 944720 840787 ZMYND11 DIP2C C10orf108 LARP5   
A06 10q11.22 47013328 47173619 160292 ANXA8      
MM648 10q11.23 51582787 51867320 284534 ASAH2 TMEM23     
MM648 10q21.1 53402176 53673530 271355 PRKG1      
D36 10q21.1 55189171 55614635 425465 PCDH15      
MM648 10q21.1 56020258 56418998 398741 PCDH15      
MM648 10q21.3 66657188 69243230 2586043 CTNNA3 LRRTM3 DNAJC12    
D05 10q21.3 67618085 67877223 259139 CTNNA3      
D03 10q21.3 67618085 68008745 390661 CTNNA3      
C-32 10q23.1 83567079 83955328 388250 NRG3      
D35 10q23.1 86071723 86272898 201176 KIAA1128      
C-32 10q23.31 89154174 89824376 670203 MINPP1 PAPSS2 ATAD1 PTEN   
A13 10q23.31 89154174 90223085 1068912 MINPP1 PAPSS2 ATAD1 PTEN C10orf59  
D36 10q23.31 89263559 90039613 776055 MINPP1 PAPSS2 ATAD1 PTEN C10orf59  
MM604 10q23.31 89408233 89610893 202661  PAPSS2 ATAD1 PTEN   
D03 10q23.31 89408233 89773113 364881  PAPSS2 ATAD1 PTEN   
MM138 10q23.31 89408233 90098699 690467  PAPSS2 ATAD1 PTEN C10orf59  
D14 10q23.31 89,509,440 90,381,995 872556   ATAD1 PTEN C10orf59  
A06 10q23.31 89574656 90098699 524044    PTEN C10orf59  
MM648 10q23.31 90175055 90451117 276063     C10orf59 to LIPF 
MM648 10q23.31 90580888 90861430 280543 ANKRD22 STAMBPL1 ACTA2 FAS   
MM466 10q24.1 97633112 97901015 267904 C10orf130 CCNJ ZNF518    
MM466 10q24.2 100367382 100915400 548019 HPSE2      
MM648 10q24.2 100500063 100915400 415338 HPSE2      
D20 10q26 131701360 132309914 608555 TXNL2      
D01 11p14.1 27304873 27410305 105433 CCDC34 LGR4     
D22 11p11.2 46188078 46701579 513502 CREB3L1 to F2     
A06 11q12.1 58004294 58358885 354592 OR5B21 LPXN ZFP91 CNTF GLYAT GLYATL2 
RPMI7932 11q14.1 79583074 80466677 883604 nil      
A06 11q22.1 98679893 99929025 1249133 CNTN5      
A06 11q22.2-q22.3 102372451 102933560 561110 DCUN1D5      
A06 11q22.3 105881574 106564323 682750 GUCY1A2      
RPMI7932 12p12.3 19055531 19442103 386573 PLEKHA5      
D20 12q21.2 75951515 76259083 307569 E2F7      
D41 12q23.1-q23.2 99757560 99931530 173971 TMEM16D      
D08 14q21.2 43684946 44415270 730325 C14orf155      
D08 14q21.2 44565513 45608748 1043236 KIAA0423 PRPF39 FKBP3 FANCM C14orf106  
MM603 15q21.1 42609135 42939663 330529 EIF3S1 KIAA1840 B2M TRIM69   
MM603 15q21.1 44300774 46093277 1792504 SEMA6D      
D36 16q13 55632716 55784457 151742 NOD27 CPNE2 NIP30 RSPRY1   
MM253 16q23.1 77062499 77252441 189943 WWOX      
MM370 16q23.1 77062499 77449191 386693 WWOX      
D24 17p13.2 5822350 6051282 228933 KIAA0523      
D24 18q21.32 56122993 56572162 449170 MC4R      
D24 18q22.1-q22.2 64447958 65009667 561710 TXNDC10 CCDC102B     
D35 20p12.1 14662457 15008369 345913 C20orf133      
WW 21q11.2-q21.1 15070648 15334458 263811 NRIP1      
A13 Xp22.33 2693518 2818764 125247 XG GYG2 ARSD    
MM548 Xp22.33-p22.2 2818764 10544773 7726010   ARSD to MID1  
HT144 Xp22.31 7056329 7894339 838011 STS VCX PNPLA4    
MM409 Xp22.2 10789307 11071711 282405 HCCS ARHGAP6 AMELX    
MM548 Xq21.32-q21.33 85312335 95600000 10287666 DACH2 to RP1-32F7.2    
MM253 Xq21.33 86196835 94633364 8436530 KLHL4 to RP1-32F7.2    
D05 Xq27.1-q27.2 139523208 142422593 2899386 CDR1 to SPANX-N3    
Cell lineCytobandStart ntEnd ntSize (nt)Genes within HD
D35 1p35.2 31140795 31691124 550330 PUM1 FAM77C WDR57 ZCCHC17 FABP3 SERINC2 
WW 1p31.2 66531644 66921109 389466 PDE4B SGIP1     
MM548 1p21.3 97732018 98107902 375885 DPYD      
D28 1q31.3 193441431 193628794 187364 CFH CFHR3 CFHR1 CFHR4   
D01 1q44 244079149 244245509 166361 OR2G3 OR13G1 OR6F1 OR5AY1   
C-32 2p25.1 7064772 7441255 376484 RNF144      
MM386 2q32.1 188169003 188683293 514291 TFPI      
D04 2q33.3-q34 212189700 212941892 752193 ERBB4      
MM603 2q37.3 239601090 239845248 244159 HDAC4      
MM426 2q37.3 239716794 240039566 322773 HDAC4      
MM473 2q37.3 239716794 240111974 395181 HDAC4      
MM370 3p14.2 60180460 60857999 677540 FHIT      
MM253 3p14.2 60200880 60697129 496250 FHIT      
D22 3q13.31 117290277 117667176 376900 LSAMP      
D35 3q25.1 152622710 153335910 713201 MED12L IGSF10 AADACL2 AADAC SUCNR1  
D10 4p16.3 63508 141601 78094 ZNF595 ZNF718     
A13 4q22 93683540 94526401 842862 GRID2      
D14 4q26 117898191 118775563 877373 TRAM1L1      
A06 5p15.1-p14.3 17916022 19862376 1946355 CDH18      
D20 5q11.1 49945313 50116451 171139 PARP8      
D64 5q11.2 55069845 55450713 380869 DDX4 IL31RA IL6ST IL6ST ANKRD55  
D64 5q11.2 56163623 56702257 538635 MGC33648 MIER3 GPBP1    
WW 6p24.1 12496151 13168554 672404 PHACTR1      
A04 6q25.3 155816691 157219651 1402961 NOX3 ARID1B     
D35 6q26 162324738 162459269 134532 PARK2      
D29 6q27 167775932 168154636 378705 LOC401286 C6orf123 MLLT4    
MM370 7q21.11 77758447 78217314 458868 MAGI2      
D29 8q11.21 51697600 52226926 529327 SNTG1      
MM384 8q12.3 64209391 65473754 1264364 YTHDF3      
D54 9p24.3 194201 1248905 1054705 C9orf66 DOCK8 ANKRD15 DMRT1 DMRT3 DMRT2 
RPMI7932 9p24.3 1463302 14264056 12800755 SMARCA2 to NFIB     
MM253 9p24.1 5170982 5520017 349036 INSL6 to PDCD1LG2    
D54 9p24.1-p23 8021935 11421917 3399983 PTPRD      
MM253 9p24.1-p23 8892753 10148149 1255397 PTPRD      
A06 9p23 9159552 9438088 278537 PTPRD      
CJM 9p23 9297216 9630335 333120 PTPRD      
D14 9p23 9565540 9910033 344494 PTPRD      
D35 9p23 10422363 13999610 3577248 PTPRD TYRP1 C9orf150 MPDZ   
D24 9p23 12892159 13169615 277457    MPDZ   
A02 9p22.3-p22.2 15750988 16629077 878090 C9orf93 BNC2     
A15 9p22.3-p22.2 16296523 16772915 476393  BNC2     
MM540 9p22.3-p22.2 16296523 16903197 606675  BNC2 LOC648570    
D35 9p22.3-p22.2 16347674 17196306 848633 C9orf39 BNC2 LOC648570    
D32 9p22.3-p22.2 16443145 17390701 947557 C9orf39 BNC2 LOC648570    
D01 9p22.1-p21.3 19791657 22197037 2405381 MLLT3 to CDKN2B    
D35 9p21.3 20316948 25319508 5002561 MLLT3 to   ELAVL2  
D54 9p21.3 20857809 21189680 331872 KIAA1797 to IFNA4     
D24 9p21.3 20903989 23401162 2497174 KIAA1797 to  DMRTA1   
MM537 9p21.3 21119246 22774110 1654865 IFNW1 to  DMRTA1   
MW 9p21.3 21119246 23297724 2178479 IFNW1 to  DMRTA1   
D29 9p21.3 21154463 25319508 4165046 IFNA21 to   ELAVL2  
CJM 9p21.3 21397250 22197037 799788 IFNA8 to CDKN2B    
HT144 9p21.3 21507302 22128823 621522 MTAP CDKN2A CDKN2B    
MM138 9p21.3-p21.2 21523610 26323942 4800333 MTAP CDKN2A CDKN2B DMRTA1 ELAVL2 TUSC1 
MM127 9p21.3 21572720 22064580 491861 MTAP CDKN2A CDKN2B    
MM418 9p21.3 21572720 22064580 491861 MTAP CDKN2A CDKN2B    
HT144 9p21.3 21572720 22182093 609374 MTAP CDKN2A CDKN2B    
D04 9p21.3 21578603 22631633 1053031 MTAP CDKN2A CDKN2B DMRTA1   
MM472 9p21.3 21673289 22280617 607329 MTAP CDKN2A CDKN2B    
D32 9p21.3 21673289 23032805 1359517 MTAP CDKN2A CDKN2B DMRTA1   
MM229 9p21.3 21690220 22064569 374350 MTAP CDKN2A CDKN2B    
MM253 9p21.3 21779250 22131886 352637 MTAP CDKN2A CDKN2B    
MM540 9p21.3 21805166 21998659 193494 MTAP CDKN2A CDKN2B    
WW 9p21.3 21805166 21998659 193494 MTAP CDKN2A CDKN2B    
A04 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
C-32 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
D40 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
HT144 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
MM418 9p21.3 21805166 22197037 391872 MTAP CDKN2A CDKN2B    
A06 9p21.3 21805166 22326954 521789 MTAP CDKN2A CDKN2B    
D08 9p21.3 21805166 22326954 521789 MTAP CDKN2A CDKN2B    
MM266 9p21.3 21805166 23243097 1437932 MTAP CDKN2A CDKN2B    
MM466 9p21.3 21805166 23432605 1627440 MTAP CDKN2A CDKN2B DMRTA1   
D49 9p21.3 21857756 22197037 339282  CDKN2A CDKN2B    
D54 9p21.3 21857756 22197037 339282  CDKN2A CDKN2B    
MM370 9p21.3 21857756 22197037 339282  CDKN2A CDKN2B    
MM648 9p21.3 21998659 22280617 281959 MTAP CDKN2A CDKN2B DMRTA1   
D22 9p21.3 21998659 23813162 1814504  CDKN2A CDKN2B DMRTA1 ELAVL2  
MM266 9p21.3 23401162 23864047 462886     ELAVL2  
WW 9p21.1 28439733 28902923 463191 LRRN6C      
WW 9p13.2 37869040 38062699 193660 MCART1 SHB     
A06 9q21.13 74224436 74417045 192610 RORB      
A13 9q31.1 101353456 101682714 329259 RNF20 GRIN3A PPP3R2    
D40 9q33.1 116760456 117221359 460904 ASTN2      
D32 9q33.2-q33.3 122191507 123462916 1271410 PTGS1 to DENND1A    
MM138 9q33.2 122382671 122817077 434407 OR1N2 to RABGAP1    
D05 10p15.3 103934 944720 840787 ZMYND11 DIP2C C10orf108 LARP5   
A06 10q11.22 47013328 47173619 160292 ANXA8      
MM648 10q11.23 51582787 51867320 284534 ASAH2 TMEM23     
MM648 10q21.1 53402176 53673530 271355 PRKG1      
D36 10q21.1 55189171 55614635 425465 PCDH15      
MM648 10q21.1 56020258 56418998 398741 PCDH15      
MM648 10q21.3 66657188 69243230 2586043 CTNNA3 LRRTM3 DNAJC12    
D05 10q21.3 67618085 67877223 259139 CTNNA3      
D03 10q21.3 67618085 68008745 390661 CTNNA3      
C-32 10q23.1 83567079 83955328 388250 NRG3      
D35 10q23.1 86071723 86272898 201176 KIAA1128      
C-32 10q23.31 89154174 89824376 670203 MINPP1 PAPSS2 ATAD1 PTEN   
A13 10q23.31 89154174 90223085 1068912 MINPP1 PAPSS2 ATAD1 PTEN C10orf59  
D36 10q23.31 89263559 90039613 776055 MINPP1 PAPSS2 ATAD1 PTEN C10orf59  
MM604 10q23.31 89408233 89610893 202661  PAPSS2 ATAD1 PTEN   
D03 10q23.31 89408233 89773113 364881  PAPSS2 ATAD1 PTEN   
MM138 10q23.31 89408233 90098699 690467  PAPSS2 ATAD1 PTEN C10orf59  
D14 10q23.31 89,509,440 90,381,995 872556   ATAD1 PTEN C10orf59  
A06 10q23.31 89574656 90098699 524044    PTEN C10orf59  
MM648 10q23.31 90175055 90451117 276063     C10orf59 to LIPF 
MM648 10q23.31 90580888 90861430 280543 ANKRD22 STAMBPL1 ACTA2 FAS   
MM466 10q24.1 97633112 97901015 267904 C10orf130 CCNJ ZNF518    
MM466 10q24.2 100367382 100915400 548019 HPSE2      
MM648 10q24.2 100500063 100915400 415338 HPSE2      
D20 10q26 131701360 132309914 608555 TXNL2      
D01 11p14.1 27304873 27410305 105433 CCDC34 LGR4     
D22 11p11.2 46188078 46701579 513502 CREB3L1 to F2     
A06 11q12.1 58004294 58358885 354592 OR5B21 LPXN ZFP91 CNTF GLYAT GLYATL2 
RPMI7932 11q14.1 79583074 80466677 883604 nil      
A06 11q22.1 98679893 99929025 1249133 CNTN5      
A06 11q22.2-q22.3 102372451 102933560 561110 DCUN1D5      
A06 11q22.3 105881574 106564323 682750 GUCY1A2      
RPMI7932 12p12.3 19055531 19442103 386573 PLEKHA5      
D20 12q21.2 75951515 76259083 307569 E2F7      
D41 12q23.1-q23.2 99757560 99931530 173971 TMEM16D      
D08 14q21.2 43684946 44415270 730325 C14orf155      
D08 14q21.2 44565513 45608748 1043236 KIAA0423 PRPF39 FKBP3 FANCM C14orf106  
MM603 15q21.1 42609135 42939663 330529 EIF3S1 KIAA1840 B2M TRIM69   
MM603 15q21.1 44300774 46093277 1792504 SEMA6D      
D36 16q13 55632716 55784457 151742 NOD27 CPNE2 NIP30 RSPRY1   
MM253 16q23.1 77062499 77252441 189943 WWOX      
MM370 16q23.1 77062499 77449191 386693 WWOX      
D24 17p13.2 5822350 6051282 228933 KIAA0523      
D24 18q21.32 56122993 56572162 449170 MC4R      
D24 18q22.1-q22.2 64447958 65009667 561710 TXNDC10 CCDC102B     
D35 20p12.1 14662457 15008369 345913 C20orf133      
WW 21q11.2-q21.1 15070648 15334458 263811 NRIP1      
A13 Xp22.33 2693518 2818764 125247 XG GYG2 ARSD    
MM548 Xp22.33-p22.2 2818764 10544773 7726010   ARSD to MID1  
HT144 Xp22.31 7056329 7894339 838011 STS VCX PNPLA4    
MM409 Xp22.2 10789307 11071711 282405 HCCS ARHGAP6 AMELX    
MM548 Xq21.32-q21.33 85312335 95600000 10287666 DACH2 to RP1-32F7.2    
MM253 Xq21.33 86196835 94633364 8436530 KLHL4 to RP1-32F7.2    
D05 Xq27.1-q27.2 139523208 142422593 2899386 CDR1 to SPANX-N3    

NOTE: For full listing of all HDs including those that do not seem to affect annotated genes, refer to Supplementary Table S1.

Figure 1.

Examples of HDs detected using Illumina 317K BeadChip SNP arrays. In each case, log2R 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 log2R. 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).

Figure 1.

Examples of HDs detected using Illumina 317K BeadChip SNP arrays. In each case, log2R 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 log2R. 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).

Close modal

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.

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).

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).

Close modal

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.

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, 2634). 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, 3436). 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 (47, 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.

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

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.

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Supplementary data