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

Research Article

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

Abstract Conventional chromosome-based comparative genomic hybridiza- Although a number of genes related to melanoma develop- tion (CGH) has been used to study melanomas of different subtypes ment have been identified through candidate gene screening (4–7), and a limited number of studies have looked at array-based approaches, few studies have attempted to conduct such CGH (aCGH) in murine (8), swine (9), and human (2, 10) mela- analyses on a genome-wide scale. Here we use Illumina 317K nomas. The latter studies have led to the identification of CDK4, whole-genome single-nucleotide polymorphism arrays to CCND1, and KIT amplifications in a subset of malignant melanoma. define a comprehensive allelotype of melanoma based on loss Although aCGH is adequate for detecting high level amplifica- of heterozygosity (LOH) and copy number changes in a panel tions and homozygous deletions (HD), it grossly underestimates of 76 melanoma cell lines. In keeping with previous reports, the level of LOH (11). In contrast, the use of high-density single- we found frequent LOH on chromosome arms 9p (72%), 10p nucleotide polymorphism (SNP) arrays has proved to be a superior approach to defining genome-wide LOH and copy number changes (55%), 10q (55%), 9q (49%), 6q (43%), 11q (43%), and 17p (41%). Tumor suppressor genes (TSGs) can be identified in a wide range of tumor types (e.g., refs. 12–20). through homozygous deletion (HD). We detected 174 HDs, Only one study to date has assessed LOH and copy number the most common of which targeted CDKN2A (n = 33). The changes in melanoma using SNP arrays (21). Eight melanoma cell second highest frequency of HD occurred in PTEN (n = 8), lines from the NCI60 series of tumor lines were analyzed using Affymetrix 100K SNP chips (Affymetrix, Santa Clara, CA). Although another well known melanoma TSG. HDs were also common 1 for PTPRD (n = 7) and HDAC4 (n = 3), TSGs recently found these data are publicly available, the authors did not present a full to be mutated or deleted in other cancer types. Analysis of summary of the results but rather focused on the key finding of other HDs and regions of LOH that we have identified might MITF amplification in some samples. lead to the characterization of further melanoma TSGs. We Here, we use high-density whole-genome SNP arrays, with an noted 197 regional amplifications, including some centered average inter-SNP spacing of 9 kb to define a comprehensive on the melanoma oncogenes MITF (n = 9), NRAS (n = 3), BRAF allelotype of melanoma based on LOH and copy number changes. (n = 3), and CCND1 (n = 3). Other amplifications potentially Tumor suppressor genes (TSG) can be identified through HD. We detected 174 HDs in a panel of 76 melanoma cell lines, the most target novel oncogenes important in the development of a subset of melanomas. The numerous focal amplifications and common of which centered on CDKN2A, PTEN, PTPRD, and HDs we have documented here are the first step toward HDAC4. The latter two loci have not previously been shown to play identifying a comprehensive catalog of genes involved in a role in melanoma development. Other HDs are likely to point to melanoma development, some of which may be useful the location of additional uncharacterized melanoma TSGs. prognostic markers or targets for therapies to treat this Oncogenes can be identified by amplification. We detected 197 disease. [Cancer Res 2007;67(6):2632–42] 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 Introduction potentially target novel melanoma oncogenes. Several genes have been shown to be mutated in melanoma, mostly through candidate gene screening approaches (reviewed in Materials and Methods ref. 1). Elucidation of such genes has important implications for defining melanoma subtypes (2) and for tailoring treatment (e.g., Cell culture and DNA extraction. The 76 melanoma cell lines used in this study were derived from primary cutaneous melanomas or melanoma MEK, KIT,orBRAF inhibitors). metastases, as described previously (22). DNA was extracted using QIAGEN Although many loss of heterozygosity (LOH) studies have been QIamp Blood Maxi kits according to the manufacturer’s instructions conducted in melanoma, most have focused on small chromosomal (Qiagen, Hilden, Germany). regions or a limited number of chromosomes. Only one study (3) Mutation analysis of cell lines. Mutations in the BRAF, NRAS, HRAS, has attempted to carry out a genome-wide allelotype, using one KRAS, CDKN2A, and PTEN genes have previously been described in this to three microsatellite markers from each chromosome arm. 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) Note: Supplementary data for this article are available at Cancer Research Online according to the manufacturer’s specifications (Illumina, San Diego, CA). (http://cancerres.aacrjournals.org/). Briefly, 750 ng of genomic DNA were amplified at 37jC overnight, using Requests for reprints: Nicholas Hayward, Oncogenomics Laboratory, Queensland solutions WG-AMM and WG-MP1. After overnight incubation, the amplified Institute of Medical Research, 300 Herston Road, Herston, Brisbane, QLD 4006, Australia. Phone: 61-7-33620306; Fax: 61-7-38453508; E-mail: [email protected]. I2007American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4152 1 http://www.ncbi.nlm.nih.gov/geo (accession number GSE2520).

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

Cell lines Site primary melanoma No. HDs No. amplicons CDKN2A PTEN BRAF NRAS

A02 non-CSD 2 0 wt wt V599E wt A04 non-CSD 2 1 HD wt wt wt A06 non-CSD 11 1 HD HD V599E wt A07non-CSD 0 4 E2K wt wt G12S A12 non-CSD 0 0 A13 non-CSD 4 3 W110Stop HD V599E wt A15 non-CSD 2 2 wt (methylated) wt V599K wt AF6 0 0 splice mutation splice mutation V599E wt C-32 non-CSD 4 5 HD HD V599E wt CJM 2 9 HD wt wt Q61K D01 occult 3 1 HD wt wt Q61K D03 non-CSD 2 0 D04 non-CSD 2 1 HD wt wt Q61L D05 non-CSD 5 1 frameshift mutation wt V599E wt D08 non-CSD 5 0 HD wt wt Q61L D10 acral 1 8 wt wt wt wt D11 mucosal 0 29 P114L wt wt Q61L D14 non-CSD 4 0 P114L HD V599E wt D17non-CSD 0 0 wt (methylated) wt V599E wt D20 non-CSD 3 16 wt HD V599E wt D22 non-CSD 6 0 HD wt wt wt D24 8 3 HD wt wt wt D25 non-CSD 0 1 wt wt V599E wt D28 occult 1 1 P114L wt V599K wt D29 4 2 HD wt V599E wt D30 non-CSD 0 0 D32 3 3 HD frameshift mutation V599E wt D35 11 0 splice mutation wt wt wt D36 non-CSD 4 0 R80Stop HD V599E wt D38 occult 0 0 wt (methylated) wt wt wt D40 non-CSD 2 1 HD wt V599E wt D41 non-CSD 1 0 wt wt V599E wt D49 non-CSD 3 0 D54 non-CSD 4 1 D64 non-CSD 2 4 HT144 4 3 HD HD V599E wt JA 0 0 wt wt V599E wt MM96 non-CSD 0 0 wt (methylated) wt V599E wt MM127non-CSD 1 0 HD wt wt G13R MM138 non-CSD 3 0 HD MM200 non-CSD 0 1 wt F56I V599E wt MM229 non-CSD 1 0 HD wt L596S wt MM253 non-CSD 6 1 HD wt V599E wt MM266 non-CSD 3 1 MM329 non-CSD 0 10 wt wt wt wt MM369 non-CSD 0 0 R80Stop wt V599E wt MM370 non-CSD 4 0 HD wt V599E wt MM384 occult 1 2 P48L wt V599E wt MM386 non-CSD 1 4 wt (methylated) HD V599E wt MM396 non-CSD 1 0 P81L wt V599E wt MM409 non-CSD 1 0 HD wt V599E wt MM415 non-CSD 1 3 wt wt wt Q61L MM418 non-CSD 2 0 HD wt V599E wt MM426 non-CSD 1 4 E88stop wt V599E wt MM466 non-CSD 6 7HD wt V599E wt MM472 CSD 2 2 HD P30L V599E wt MM473 non-CSD 2 2 H83Y wt V599E wt MM485 non-CSD 0 5 W110Stop wt wt Q61R

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Table 1. Summary of melanoma cell lines and mutation status of selected oncogenes and TSGs (Cont’d)

Cell lines Site primary melanoma No. HDs No. amplicons CDKN2A PTEN BRAF NRAS

MM488 non-CSD 0 2 HD HD V599E wt MM537non-CSD 2 0 HD wt V599E wt MM540 non-CSD 2 0 wt (methylated) wt V599E wt MM548 non-CSD 3 1 delR99 wt V599E wt MM595 non-CSD 1 6 HD wt V599E wt MM603 3 2 wt wt V599E wt MM604 non-CSD 1 11 wt HD V599E wt MM622 non-CSD 0 0 frameshift mutation L139Stop V599E wt MM628 occult 0 1 P114L wt wt G13R MM636 non-CSD 0 1 MM647non-CSD 0 0 aberrant transcript wt wt wt MM648 non-CSD 10 1 HD wt V599E wt MM649 non-CSD 0 2 wt (methylated) wt V599E wt MW 1 7HD wt V599R wt RPMI7932 3 4 SKMEL28 1 9 wt T167A V599E wt SKMEL5 0 6 HD del exon 1 V599E wt WW 6 3 HD wt V599E wt

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

DNA was fragmented using WG-FRG and precipitated with isopropanol after cell line derived from an acral melanoma is wild-type for all of the the addition of WG-PA1. The dried precipitated pellet was then resuspended above genes. The cell line derived from a mucosal melanoma carried in WG-RA1 and hybridized to a beadchip along with WG-RA1 and a missense mutation affecting CDKN2A (P114L) and an NRAS j formamide. The arrays were then incubated overnight at 48 C, after which mutation (Q61L). they underwent single-base extension on a Teflow chamber rack system Whole chromosome arm copy number aberrations. Com- (Tecan, Ma¨nnedorf, Switzerland) using WG-XC1, WG-XC2, and WG-TEM. After the single-base extension step, the beadchips were stained with bined LOH/CGH revealed a number of recurrent copy number WG-LTM and WG-ATM, dried for 1 h, then imaged using a BeadArray Reader changes affecting whole chromosome arms (Table 2). Most (Illumina). Image data was analyzed using Beadstudio 2.0 (Illumina). All common among the losses were both arms of chromosomes 9 genomic positions were based upon hg17from the UCSC Genome Browser. 2 and 10, which occurred in f40% to 50% of all samples. The For additional details and example outputs, refer to Peiffer et al. (25). majority of these losses were due to hemizygous deletions, but 30% to 40% were due to copy number neutral LOH or a combination of Results copy number neutral LOH and hemizygous deletion. The next most TSG and oncogene mutation status. Table 1 summarizes the frequent were losses of 17q (30%) and 17p (25%). Approximately status of several key oncogenes and TSGs in the panel of 76 mela- 75% of the latter were the result of copy number neutral LOH, noma cell lines. At least 54 (71%) had CDKN2A defects, 48 (63%) had which contrasted with the 40% of losses on the q arm that occurred BRAF mutations, 10 (13%) had NRAS mutations (none had muta- through this mechanism. The only other chromosome arm tions in HRAS or KRAS), and 17(22%) had PTEN defects. No cell line exhibiting complete loss in >20% of samples was 5q (24%), half had a concomitant defect in NRAS and BRAF,orNRAS and PTEN. of which were due to hemizygous deletion (Table 2). All cell lines with a PTEN defect also carried a BRAF mutation. The most common copy number gains occurred for chromo- We assessed the site or histotype of the primary melanomas in the some arms 2p, 6p, 7p, 7q, 19q, 20p, 20q, and 22q. Each were present patients from whom the cell lines were derived. For 19 of the cell in 20% to 30% of the cell lines. Trisomy for these chromosome arms lines, the site of the primary lesion was unknown; this included five was the most common copy number deviation, although the occa- patients who presented with metastatic disease and had no sional cell line was found to harbor up to eight copies (Table 2). detectable primary tumor (i.e., occult primary). Based on the Regional LOH analysis. The whole-genome SNP data were categorization of Curtin et al. (2), 54 (95%) of the remaining 57lines further interrogated for regions of LOH that occurred across were derived from nonchronic sun-damaged lesions, one was from a smaller intervals than a whole chromosome arm. Cross-referencing chronic sun-damaged site (the nose), one was an acral (plantar) the Database of Genomic Variants3 was done to eliminate calling melanoma, and one was from a buccal mucosa melanoma (Table 1). common structural polymorphisms as small regions of LOH. Thus, although the bulk of our data reflect nonchronic sun-damaged Table 3 lists the cytobands and nucleotide intervals showing melanomas, the inclusion of isolated examples of melanomas of the most frequent regional LOH on each chromosome arm. The other histotypes allows some salient comparisons to be made. The commonest occurred at 9p21.3 (position 20317754–25227815) and

2 http://genome.ucsc.edu/ 3 http://projects.tcag.ca/variation/

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Genome-Wide LOH and Copy Number Analysis in Melanoma targeted the CDKN2A locus (72%). The next most frequent (55%) Mb. Table 4 lists the chromosomal regions showing HD in one or were LOH of 10q23.2-q21.33 (position 87491745–87930977), target- more cell lines and the genes that reside in these regions. The full ing PTEN, and 10pter-p15.1 (position 103934–5767329), targeting list of all HDs, including those that did not affect annotated genes, an as yet unidentified TSG. Overall levels of LOH of >40% also is given in Supplementary Table S1. On average, each cell line occurred at 6q25.1-q25.3, 9q21.2-q21.33, 11q22.3, and 17p13.1. carried 2.3 HDs (range, 0–11; Table 1). Notably, the mucosal and Homozygous deletions. In the panel of 76 melanoma cell lines, acral melanoma cell lines were among those with the lowest we detected a total of 174 HDs. Of these, 52 HDs seemed to target frequency of deletions, having zero and one HD, respectively. a single locus, 87(50%) targeted more than one gene, and 35 of the Sixteen of the HDs occurred in more than one cell line (Table 4). HDs did not encompass the coding region of an annotated gene The most common HD (43%, 33 of 76 cell lines) affected CDKN2A (human genome build hg17). HDs ranged in size from 78 kb to 12.8 (Fig. 1A) and extended to variable numbers of neighboring genes

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

c Chromosome Complete loss Partial loss* Gains arm b b b b b b Hemi Neut Mixed Total Hemi Neut Mixed Total 3 4 5 6 7 8 Total

1p 5 2 0 7(9) 1 7 3 11 (14) 1 3 4 (5) 1q 1 5 0 6 (8) 0 11 2 13 (17) 10 1 2 13 (17) 2p 1 6 1 7(9) 1 16 0 17(22) 8 4 2 1 15 (20) 2q 0 6 1 7(9) 1 13 0 14 (18) 4 1 2 7(9) 3p 1 1 1 3 (4) 1 11 1 13 (17) 2 2 1 5 (7) 3q 0 0 2 2 (3) 1 14 3 18 (24) 2 2 4 (5) 4p 3 4 0 7(9) 0 19 3 24 (32) 2 1 1 4 (5) 4q 3 2 1 6 (8) 1 15 3 19 (25) 0 (0) 5p 4 6 0 10 (13) 0 19 1 20 (26) 6 2 8 (11) 5q 9 5 4 18 (24) 1 14 0 15 (20) 1 1 2 (3) 6p 0 4 1 5 (6) 0 18 0 18 (24) 11 3 1 15 (20) 6q 5 2 2 9 (12) 2 10 1 13 (17) 0 (0) 7p 1 2 2 5 (6) 0 7 0 7 (9) 15 2 3 20 (26) 7q 0 1 4 5 (6) 0 4 0 4 (5) 12 2 3 1 18 (24) 8p 6 4 0 10 (13) 0 19 1 20 (26) 2 1 1 4 (5) 8q 0 2 0 2 (3) 0 16 1 17(22) 8 1 1 1 11 (14) 9p 20 10 4 34 (45) 2 5 1 8 (11) 0 (0) 9q 19 11 3 33 (43) 1 5 2 8 (11) 1 1 (1) 10p 24 6 6 36 (47) 1 3 2 6 (8) 1 (1) 10q 276 6 39 (51) 1 3 3 7(9) 0 (0) 11p 9 2 1 12 (16) 2 24 0 26 (34) 0 (0) 11q 72 1 10 (13) 2 15 0 17(22) 0 (0) 12p 1 2 0 3 (4) 0 14 8 22 (29) 8 8 (11) 12q 1 3 2 6 (8) 0 13 8 21 (28) 5 5 (7) 13q 4 3 3 10 (13) 4 6 3 13 (17) 4 4 (5) 14q 8 1 0 9 (12) 4 5 10 19 (25) 0 (0) 15q 1 2 2 5 (6) 0 9 2 11 (14) 4 2 6 (8) 16p 0 2 0 2 (3) 1 18 0 19 (25) 6 1 1 8 (11) 16q 5 3 0 8 (11) 1 12 1 14 (18) 4 1 5 (7) 17p 5 14 0 19 (25) 0 3 0 3 (4) 12 1 13 (17) 17q 14 9 0 23 (30) 0 3 0 3 (4) 7 7 (9) 18p 8 5 0 13 (17) 2 13 5 20 (26) 1 1 (1) 18q 0 1 0 1 (1) 712 5 24 (32) 0 (0) 19p 0 5 1 6 (8) 1 3 0 4 (5) 10 1 11 (14) 19q 0 4 0 4 (5) 0 5 0 5 (7) 12 2 1 15 (20) 20p 0 5 0 5 (6) 0 5 0 5 (7) 12 1 2 15 (20) 20q 0 1 0 1 (1) 0 0 0 0 (0) 14 1 3 1 19 (25) 21q 3 0 1 4 (5) 0 18 2 20 (26) 2 1 3 (4) 22q 0 5 0 5 (6) 1 0 0 1 (1) 20 2 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). cOnly 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. bNumber of chromosome arms.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research on 9p22-p21, including CDKN2B, DMRTA1, ELAV2, the IFN cluster, mucosal melanoma (Table 1). Supplementary Table S2 lists the KIAA1797, MTAP, MLLT3, PTPLAD2, and TUSC1 (Table 4). The chromosomal regions showing focal amplification in one or next most common deletions affected the PTEN (Fig. 1B), PTPRD more cell lines and the genes that reside in these regions. With (Fig. 1C), and HDAC4 (Fig. 1D) loci and occurred in eight (11%), a few exceptions, including MITF (Fig. 2A), BRAF, CDH9, seven (9%), and three (4%) cell lines, respectively. With the DKFZp564N2472, GBE1, LRFN5, NAALADL2, NFIB, PTPRD, SESN3, exception of one intragenic microdeletion in PTEN we reported SLC26A7, SNTG1, TMEFF2, and WDR72, the amplicons contained previously (24), there was complete correlation between previous multiple genes (range, 1–131). Several recurring amplifications CDKN2A and PTEN deletions detected by PCR sequencing (23, 24) seemed to target the same genes (Supplementary Tables S2 and and those detected here by aCGH. S3). The most common of these centered on MITF and was seen in Regional amplifications. There were a total of 197focal (i.e., nine (12%) samples. Others included genes such as ANK1 (Fig. 2B). subchromosomal arm) amplifications in the panel of melanoma Rare instances of focal amplification, including the BRAF, CCND1, cell lines. These were generally much larger than the HDs and MDM2, NRAS, and PIK3CA genes, were also observed (Supple- ranged in size from 228 kb to 28.8 Mb. The number of amplicons mentary Table S3). Notably, none of these amplifications of docu- per cell line ranged from 0 to 29, with the most occurring in the mented melanoma oncogenes occurred in the acral or mucosal

Table 3. Most common regional LOH intervals

Chromosome SRO within Start base End base Start base End base n Total (%) arm cytoband(s) position position position position

1p 31.1 816884078445590 13 20 (26) 1q 42.2 228366540 228914590 6 12 (16) 2p 25.2 and 24.3 5805898 6119949 15755054 16984942 5 12 (16) 2q 37.3 239293533 242759899 12 19 (25) 3p 21.31 4595098749696536 14 17(22) 3q 26.31 176577059 176998125 10 12 (16) 4p 15.1 32418953 33463430 8 15 (20) 4q 32.1 and 32.3 157984245 158621187 164832073 166472504 10 16 (21) 5p 14.3 22699011 23194868 6 16 (21) 5q 11.2 58447527 58681562 7 25 (33) 6p 22.2-21.33 29361735 32298598 3 8 (11) 6q 25.1-25.3 152362786 155321679 24 33 (43) 7p 21.3-21.1 7187594 17555849 3 8 (11) 7q 22.3-31.1 105769871 111045610 8 13 (17) 8p 12 31614916 32931772 5 15 (20) 8q 11.21-11.22 and 21.3 48136481 51946973 89271300 90686126 3 5 (7) 9p 21.3 20317754 25227815 21 55 (72) 9q 21.2-21.33 77651737 83134614 4 37 (49) 10p ter-15.1 103934 5767329 6 42 (55) 10q 23.2-23.31 87491745 87930977 3 42 (55) 11p 14.1 and 11.2 29001470 29431689 45266986 46642836 9 21 (28) 11q 22.3 102626683 105738304 23 33 (43) 12p 12.1 20781209 21633573 7 10 (13) 12q 21.32 and 24.31 87076009 87733383 119303242 123506991 10 16 (21) 13q 33.3-34 108060380 114108121 717(22) 14q 32.2-ter 98443619 1063450976 15 (20) 15q 21.1 46980083 47525525 11 16 (21) 16p 12.1 23645856 24292120 5 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 7 30 (39) 18p 11.32 59836 12655072 15 (20) 18q 22.1 and 22.3 62097585 65010233 67876215 68301919 6 7 (9) 19p 13.3 217034 1822341 6 12 (16) 19q 13.32 52070865 53777522 6 10 (13) 20p 13 and 12.3 1110370 3654863 6717533 8499344 7 12 (16) 20q 13.2 52853092 53531064 3 4 (5) 21q 21.1 19744931 21665674 13 17 (22) 22q 12.2 29897517 30554739 7 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.

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Table 4. Summary of HDs that include an annotated gene

Cell line Cytoband Start nt End nt Size (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 114219173399983 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 16903197606675 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

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Table 4. Summary of HDs that include an annotated gene (Cont’d)

Cell line Cytoband Start nt End nt Size (nt) Genes within HD

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 232430971437932 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 22280617281959 MTAP CDKN2A CDKN2B DMRTA1 D22 9p21.3 21998659 23813162 1814504 CDKN2A CDKN2B DMRTA1 ELAVL2 MM266 9p21.3 23401162 23864047462886 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 122191507123462916 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 90381995 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

(Continued on the followingpage )

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Table 4. Summary of HDs that include an annotated gene (Cont’d)

Cell line Cytoband Start nt End nt Size (nt) Genes within HD

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 1466245715008369 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.

melanoma cell lines. Among a large number of focal amplifications 33% LOH on 5q, a chromosome not previously associated with in the acral melanoma were amplifications that included MAP3K8 harboring a putative TSG for melanoma. (Fig. 2C) and SMO (Supplementary Table S2). The numerous The SNP arrays were very effective at detecting HDs. Exemplifying amplifications seen in the mucosal melanoma included CCNB2 the power of this approach, we ‘‘rediscovered’’ the high frequency of and TERT. HDs of CDKN2A and PTEN in melanoma (Fig. 1A and B). These Although we detected high-level focal amplification of the NRAS genes were also the targets of the most recurrent regions of focal gene (e.g., Fig. 2D) in two cell lines that carried NRAS point LOH (Table 3). We have also identified many regions of HD that mutations (A07and CJM), there was generally poor correlation putatively target other TSGs. In keeping with the analysis of other between copy number increases of BRAF, HRAS, KRAS, and NRAS, cancer genomes (37), we have also found that some HDs target and concomitant mutations in these oncogenes. There were no fragile sites. We observed HDs in both the FHIT (FRA3B) and focal amplifications affecting CDK2, CDK4, CDK6, HRAS, KRAS, KIT, WWOX (FRA16D) loci in two cell lines (Table 4). Some of the genes or MYC. 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 Discussion breast cancer, and PTPRD and GUCY1A2, both listed as candidate Here we provide the first comprehensive whole-genome cancer gene for colon cancer. This suggests that these genes may allelotype for melanoma. Whole chromosome arm LOH was most also be the targets of the deletions in melanoma and imply that common on 9p, 9q, 10p, and 10q and occurred in f40% to 50% of the genes might be general TSGs affecting tumorigenesis in a all samples (Table 2). Of these 30% to 40% were due to copy wide variety of cancer types. Similarly, we found other loci number neutral LOH, which would be missed by conventional deleted in the melanoma cell line panel that belong to a number metaphase spread or BAC aCGH. When focal LOH was also taken of gene families with members mutated in breast and colon into account (Table 3), the same four chromosome arms showed cancers (38), for example, CD274, CDH18, CNTN5, DDX4, GRIN3A, the highest overall frequencies of LOH (49–72%) in keeping with KCNV2, LGR4, LRRN6C, MAGEC1, MAGEC2, MAGEC3, PCD11X, previous reports (e.g., refs. 3, 26–34). Overall LOH was next most PCDH15, PLEKHA5, PRPF39, RFX3, SEMA6D, SLC1A1, and ZFP91. common (>40%) on 6q, 11q, and 17p, once again, in support of This supports a role for these gene families as general tumor prior studies (e.g., refs. 3, 30, 31, 34–36). Additionally, we observed suppressors. www.aacrjournals.org 2639 Cancer Res 2007; 67: (6). March 15, 2007

Numerical chromosome copy number increases, and focal the second amplification peak centered on the MDM2 gene. None amplifications putatively target oncogenes. We found the most of the three samples with amplification of either gene had defects common (z25%) whole chromosome arm copy number gains in CDKN2A (affecting either p16 or p14), indicating that ampli- occurred for 7p, 20q, and 22q (Table 2). This concurs with the fication of the CDK4 and MDM2 genes is an alternative way for conventional chromosome CGH data for these chromosome arms tumor cells to simultaneously inactivate the pRb and p53 pathways. in various melanoma subtypes (4–7, 39). Regional amplifications of We found a regional amplification of MDM2 (that did not extend to KIT, MITF, BRAF, NRAS, HRAS, KRAS, MDM2, CDK4, CCND1, MYC, encompass CDK4) in a single cell line, MM595, in which the entire and PIK3CA have been reported in some melanomas. Here we CDKN2A locus is homozygously deleted. found amplification of MITF in nine cell lines (12%) in keeping with CCND1 is also a commonly amplified locus in melanoma, the frequency of 10% in primary melanomas and 15% to 20% in occurring most frequently in the acral subtype (2). This locus was melanoma metastases reported previously (21). KIT amplifications amplified in three of our melanoma cell lines (A06, MM548, and and/or mutations have been found in 28% to 39% of acral, mucosal, SKMEL5). PIK3CA mutations have been found in 1 of 118 primary and chronic sun-damaged melanomas but occur very rarely in melanomas and 1 of 34 melanoma cell lines analyzed (43), as well nonchronic sun-damaged melanomas (10, 40, 41). In support of the as in secondary melanomas from 2 of 87patients tested (44). Only latter finding, we saw no amplifications of KIT in our panel of one of the melanoma cell lines studied here carried a PIK3CA predominantly nonchronic sun-damaged cell lines. CDK4 amplifi- mutation (D17; ref. 43), but because PIK3CA activation can also cations were found in 11 (9%) samples (six acral, four mucosal, and occur through gene amplification (45), we assessed whether regio- one chronic sun-damaged melanomas) in a study of 126 mela- nal amplifications of this locus were evident from the aCGH data. nomas of various histologic types (2). In another report, CDK4 One cell line was observed to have PIK3CA amplification (MM636). amplification was documented in 3 (6%) of 51 melanoma meta- Others have reported increased copies of MYC, on chromosome stases (42). In two of these tumors, the amplicon was bipartite, with arm 8q, in 15 (37%) of a series of 41 primary or secondary

Figure 1. Examples of HDs detected using Illumina 317K BeadChip SNParrays. In each case, log 2 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).

Cancer Res 2007; 67: (6). March 15, 2007 2640 www.aacrjournals.org

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). melanomas analyzed by fluorescence in situ hybridization (46). In Moreover, some of these may prove to be useful clinically, either contrast, we did not observe focal amplifications of MYC in any cell as prognostic markers or as targets for therapies to treat line, and only 14% of samples had increased copies of 8q (Table 2). melanoma. No regional amplifications of the above-mentioned genes were seen in either the acral or mucosal cell line in our panel. However, both of Acknowledgments these lines carried a large number of amplifications at other Received 11/9/2006; revised 1/4/2007; accepted 1/9/2007. chromosomal sites. Indeed, the mucosal melanoma possessed the Grant support: National Health and Medical Research Council of Australia largest number of focal amplifications (n = 29), supporting previous (NHMRC), Queensland Cancer Fund, and Senior Principal Research Fellowship of NHMRC (N. Hayward). observations (2). The costs of publication of this article were defrayed in part by the payment of page The large number of amplifications and HDs we have charges. This article must therefore be hereby marked advertisement in accordance documented here provides a platform for further studies aimed with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Jane Palmer, Sandra Pavey, and Cathy Lanagan for tracking clinical at identifying novel melanoma oncogenes and TSGs and details of the patients and the groups of Peter Parsons and Chris Schmidt for their understanding how they contribute to melanoma development. efforts in establishing the majority of melanoma cell lines used in this study.

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Mitchell Stark and Nicholas Hayward

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