Chromosome 3P12.3-P14.2 and 3Q26.2-Q26.32 Are Genomic Markers for Prognosis of Advanced Nasopharyngeal Carcinoma

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Chromosome 3p12.3-p14.2 and 3q26.2-q26.32 Are Genomic Markers for Prognosis of Advanced Nasopharyngeal Carcinoma

Jim Jinn-Chyuan Sheu,1,4 Chia-Huei Lee,6 Jenq-Yuh Ko,7,8 George S.W. Tsao,11 Chung-Chun Wu,6 Chih-Yeu Fang,6 Fuu-Jen Tsai,2,5 Chun-Hung Hua,3 Chi-Long Chen,10 and Jen-Yang Chen6,9 Departments of 1Medical Research, 2Medical Genetics, and 3Otolaryngology, China Medical University Hospital, 4Graduate Institute of Chinese Medical Science, and 5School of Post-baccalaureate Chinese medicine, China Medical University, Taichung, Taiwan; 6National Institute of Cancer Research, National Health Research Institutes, Zhunan, Taiwan; 7Department of Otolaryngology, National Taiwan University Hospital, 8Department of Otolaryngology and 9Graduate Institute of Microbiology, College of Medicine, National Taiwan University, and 10Department of Pathology, Taipei Medical University, Taipei, Taiwan; and 11Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong SAR, China

Abstract

Purpose: Nasopharyngeal carcinoma is an epithelial p23. Six candidate genes, GPR160 (3q26.2-q27), SKIL malignancy with a remarkable racial and geographic (3q26), ADAMTS9 (3p14.2-p14.3), LRIG1 (3p14), MPDZ distribution. Previous cytogenetic studies have shown (9p22-p24), and ADFP (9p22.1) were validated by quan- nasopharyngeal carcinoma to be characterized by gross titative PCR. Fluorescence in situ hybridization studies genomic aberrations. However, identification of suscep- revealed amplification of GPR160 (in 25% of cases) and tible gene loci in advanced nasopharyngeal carcinoma SKIL (33%); and deletion of ADAMTS9 (30%), LRIG1 has been poorly discussed. (35%), MPDZ (15%), and ADFP (15%). Clinical associa- Experimental Design: A genome-wide survey of gene tion analyses indicated a poor survival rate with genetic copy number changes was initiated with two nasopha- alterations at the defined 3p deletion (P = 0.0012) and ryngeal carcinoma cell lines by array-based comparative the 3q amplification regions (P = 0.0114). genomic hybridization analysis. These alterations were Conclusion: The combined microarray technologies confirmed by a parallel analysis with the data from the suggested novel candidate oncogenes, amplification gene expression microarray and were validated by of GPR160 and SKIL at 3q26.2-q26.32, and deletion quantitative PCR. Clinical association of the defined of tumor suppressor genes ADAMTS9 and LRIG1 at target genes was analyzed by fluorescence in situ 3p12.3-p14.2. Altered expression of these genes may hybridization on 48 metastatic tumors. be responsible for malignant progression and could Results: A high percentage of genes were consistently be used as potential markers for nasopharyngeal car- altered in dosage and expression levels with gain on cinoma. (Cancer Epidemiol Biomarkers Prev 2009;18 3q26.2-q26.32 and losses on 3p12.3-p14.2 and 9p21.3- (10):2709–16)

Introduction

Nasopharyngeal carcinoma is a malignant tumor of epi- compared with Caucasians (8), suggest genetic predispo- thelial origin. As one of the most common cancers in sition may play a significant role in the onset of nasopha- southern China and Southeast Asia (1), it causes a high ryngeal carcinoma. The aggravation of genomic incidence of fatality among the inhabitants of endemic re- aberrations signifies poor prognosis in nasopharyngeal gions. Genetic susceptibility (2-4), EBV infection (5), and carcinoma patients (9, 10), and the degree of genome in- chemical carcinogens (6, 7) are thought to be implicated stability is directly related to the invasiveness and tumor- in the pathogenesis of nasopharyngeal carcinoma. igenesis found for some nasopharyngeal carcinoma cell The remarkable racial distribution and the much higher lines (11, 12). These results clearly indicate the profound risk of nasopharyngeal carcinoma in US-born Chinese, influence of genetic aberrations on relapse and metastasis of this malignancy. Nasopharyngeal carcinoma is characterized by gross Received 4/13/09; revised 6/19/09; accepted 7/27/09; published OnlineFirst 9/22/09. numerical abnormalities of chromosomes, which fre- Grant support: Grants from National Health Research Institutes (97A1CAPP18-014; quently would result in the altered expressions of onco- 98A1CAPP13-014), China Medical University Hospital (CMU96-269), and the National Science Council, Taiwan (NSC96-3112-B-400-010; NSC97-3112-B-400-007). genes and tumor suppressor genes. Conventional and Note: Supplementary data for this article are available at Cancer Epidemiology molecular cytogenetic methods, such as comparative Biomakers and Prevention Online (http://cebp.aacrjournals.org/). genomic hybridization (CGH) analysis and loss of hete- J.J.-C. Sheu and C.-H. Lee contributed equally to this work and should be considered rozygosity studies have located multiple regions of chro- joint first authors. mosomal gains and losses in nasopharyngeal carcinoma. Requests for reprints: Jen-Yang Chen, National Institute of Cancer Research, National Health Research Institutes, R2, R1211, Keyan Road, Zhunan Town, Miaoli County 350, Gains of 1q, 2q, 3q, 4q, 6p, 7q, 8q, 11q, 12p, 12q, 15q, Taiwan. Phone: 886-37-246-166, ext. 35123; Fax: 886-37-586-402. E-mail: [email protected] 17p, 17q, and 20q, and losses of 1p, 3p, 8p, 9p, 11q, 13q, and Chi-Long Chen, 111, Section 3, Hsing-Long Road, Taipei City 116, Taiwan. Phone: 886-2-2930-7930, ext. 1480; Fax: 886-2-8662-1138. E-mail: [email protected] 14q, and 16q have been found to occur with high frequen- Copyright © 2009 American Association for Cancer Research. cy in tumor samples and cell lines (13-19). In addition, doi:10.1158/1055-9965.EPI-09-0349 evolutionary tree models for this malignancy have been

Cancer Epidemiol Biomarkers Prev 2009;18(10). October 2009

constructed based on CGH data. Loss of 3p and gain of samples was verified using the RNA 6000 Nano Assay 12p are important early events, and gain of 8q is a late (Agilent Technologies). event, in nasopharyngeal carcinoma progression (20, 21). Nevertheless, the limited resolution of the tech- Array-Based CGH Analysis. A microarray containing nologies precludes the identification of candidate genes. >244,000 oligonucleotide probes designed to cover cod- Thus, the molecular mechanism underlying carcinogene- ing, noncoding, and intergenic sequences in the human sis of nasopharyngeal carcinoma remains obscure. genome (Human Genome CGH microarray 244A, Agilent Following the recent development of array-based CGH Technologies) was used for array-CGH analysis. Genomic with higher mapping resolution, some studies successful- DNA fragmentation, labeling, and array hybridization ly defined the regions of genomic imbalance to the cyto- were done according to the standard array CGH protocol band level. Deletions in 3p13-p23, 11q14-q23, 13q12-q31, (version 4) provided by Agilent Technologies as described and 14q24-qter, and amplifications of 1p34.3, 1q25-qter, previously (12, 28). For each array-CGH experiment, we used DNA isolated from nasopharyngeal carcinoma cell 3q26, 12p13, and 12q13 were detected in nasopharyngeal – carcinoma genomes (22-24). However, the power to yield lines as the experimental genome (cyanine 5 labeled) and the commercial healthy Caucasian male genome (Pro- meaningful information was impaired in previous studies – by either a lack of clinical samples or failure to validate mega) as the reference (cyanine 3 labeled). The hybridized arrays were scanned immediately at a the array results by other technologies. Moreover, the μ bacterial artificial chromosome array for identification of resolution of 5 using an Agilent G2565BA DNA micro- candidate genes remains problematic because several array scanner. The microarray images were analyzed us- genes may be identified with a single probe. ing Agilent Feature Extraction software, version 8.1.1. The purpose of our study was to define the high- Another custom analytical software package, Agilent resolution regions of gains and loss in nasopharyngeal CGH Analytics, version 3.4, was used for the subsequent carcinoma, and through integration of copy number and data analysis. The locations of the copy number aberra- gene expression profiles, to locate the putative onco- tions were calculated using the Aberration Detection genes/tumor suppressor genes that may be involved in Method 2 (ADM2) statistical algorithm. The ADM2 tumorigenesis. In addition, we also aimed to evaluate threshold was set at 9.0 to make an amplification or dele- the changes of gene dosage associated with clinical cases. tion call. According to these settings, the aberration score For that goal, we carried out a genome-wide survey of was generated automatically for each altered locus. the consistent changes in DNA copy number and expres- Gene Expression Microarray Analysis. The whole sion levels throughout the genomes of nasopharyngeal human genome oligo microarray kit (Agilent Technolo- carcinoma cell lines, using array-based CGH and expres- gies) and Agilent's low RNA input linear amplification/ sion profiling experiments. Instead of a bacterial artificial one-color labeling kit were used for gene expression chromosome array, the use of oligoarray (average spatial microarray analysis, all of which were conducted in bio- resolution approximately 7 kb with multiple probes for logical duplicate. Target preparation and array hybridiza- each gene) for array CGH has the potential to indicate – tion essentially followed the protocol recommended by the nasopharyngeal carcinoma associated genes directly. Agilent Technologies. Briefly, 1,000 ng of total RNA were To evaluate the clinical prevalence, the most significant reverse transcribed into cDNA using the T7 promoter alterations found were validated in tissue microarray primer. Cyanine 3-dCTP–labeled cRNA was synthesized using gene-specific fluorescence in situ hybridization from the cDNA by using the T7 RNA polymerase. To re- (FISH). Sequentially we also examined the recurrent move unincorporated nucleotides, the labeled cRNA was copy-number changes associated with patients' survival purified using an RNeasy mini kit (Qiagen). Array hy- rate. The results may provide a starting point for conduct- bridization was done in 100 μL of a hybridization mixture ing further investigations by characterizing the roles of containing cRNA probes at 65°C for 17 h. possible target genes found in this study. The hybridized arrays were then washed and scanned using an Agilent G2565BA DNA microarray scanner. Fluorescence intensities of spots were quantified, back- Materials and Methods ground-subtracted, and dye-normalized by Feature Ex- traction software (Agilent Technologies). Raw data were Cell Lines. Three established human nasopharyngeal imported and analyzed using GeneSpring GX (Agilent cell lines were investigated in this study. NPC-TW01 Technologies) to generate gene lists of differentially ex- was obtained from a moderately differentiated nasopha- pressed (change in expression with fold change >2). ryngeal carcinoma specimen, keratinizing squamous cell Quantitative Real-Time PCR. We designed PCR pri- carcinoma (25). HONE1 was derived from poorly differ- mers for the potential candidate genes within the defined entiated squamous cell carcinoma of the nasopharynx aberration regions using the primer 3 program (http:// (26). NP460hTert is a telomerase-immortalized naso- www.genome.wi.mit.edu/cgi-bin/primer/primer3_ pharyngeal epithelial cell line with an almost diploid www.cgi; Supplementary Table 1). Quantitative real time karyotype (27). All cells were cultured as described PCR was done on the cDNA extracted from NPC-TW01 previously. and HONE1 cells. The cDNA from NP460hTert cells was DNA and RNA Extraction. Genomic DNA was ex- used as the normal control for comparison. PCR reactions tracted using the DNeasy tissue kit (Qiagen) according were done in triplicate and threshold cycle numbers were to the manufacturer's instructions, as described previ- calculated using the iCycler software v2.3 (Bio-Rad Labo- ously (12, 28). Total RNAs were extracted with an ratories). Expression levels of candidate genes within each RNeasy mini kit (Qiagen) according to the manufac- sample were determined by normalization to the expres- turer's instruction. The integrity of the DNA and RNA sion level of GAPDH.

Cancer Epidemiol Biomarkers Prev 2009;18(10). October 2009

Tissue Microarray for FISH. We arranged 48 formalin- including tumor site, clinical stage, treatment history, fixed, paraffin-embedded metastatic nasopharyngeal car- recurrent status, and survivorship. Overall survival time cinoma tissues and two normal oral epithelia onto tissue was defined as the number of months between diagnosis microarrays to facilitate FISH. Four representative cores and death or between diagnosis and the most recent from each tumor were placed on the tissue microarrays. follow-up. All calculations and statistical analyses were Acquisitions of tissue specimens and patient information carried out using the SAS/STAT software package (SAS were approved for this study by the Institutional Review Institute) and plotted as survival curves using the Board at Taiwan University Hospital. Bacterial artificial Kaplan-Meier method. With this method, P values were chromosome clones, RP11-257J13 for ADAMTS9,CTD- evaluated by log-rank test. 2111L7 for LRIG1, CTD-3181O10 for GPR160,CTD- 2320D24 for SKIL, RP11-806B4 for MPDZ,and RP11-909I1 for ADFP, were purchased from Invitrogen. Results Bacterial artificial chromosome clone RP11-61K12 was uti- Impact of Copy Number Alterations on Gene Expres- lized as the universal control probe for all the genes tested sion. Global copy number aberrations were analyzed because no copy number change was found within this using high-resolution oligoarray CGH on two nasopha- region based on our array CGH data. The method used ryngeal carcinoma cell lines, NPC-TW01 and HONE1, for FISH has been described in a previous study (12). which were originally defined as metastatic nasopharyn- Two individual scientists who were not aware of the tu- geal carcinoma. The overviews of array CGH profiles re- mor grade and clinical information evaluated FISH sig- vealed high similarity between the nasopharyngeal nals. Approximately 100 tumor cells were examined carcinoma cell lines (Fig. 1 and Supplementary Table S2), from each specimen. Gene amplification was defined as but the degree of copy-number alterations was more a ratio of gene probe signal to control probe signal >2.8. severe in NPC-TW01 than in HONE1. There were 1,204 Gene deletion was defined as a negative ratio of control and 1,513 genes with copy-number gain (copy number probe signal to gene probe signal <-2.0. variations with aberration score >0.5 and number of con- Statistical Analysis and Clinical Correlation. The clin- tiguous probes ≥3) in NPC-TW01 and HONE1, respec- ical information of nasopharyngeal carcinoma samples tively. Among them, 850 were amplified in both cell studied in this study was collected from clinical records, lines (Gain-TH). There were 3,525 genes and 926 genes

Figure 1. Whole genomic copy-number profiles of nasopharyngeal carcinoma cell lines TW01 and HONE1. The array CGH profile is shown adjacent to the ideogram of each chromosome. The results show the log2 value of the normalized signal intensity ratio from the array experiment. A log2 signal ratio of zero represents no aberrant copy number in TW01 and HONE1. Where log2 >0, it suggests amplified DNA copy-number for TW01/HONE1 relative to the reference human male genome. Where log2 <0, the reverse is inferred. Images were produced using CGH Analytics version 3.5.14 software. The array CGH data discussed in this publication have been deposited in the National Center for Biotechnology Information's Gene Expession Omnibus (50) and are accessible through GEO series accession number GSE15172 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15172).

Cancer Epidemiol Biomarkers Prev 2009;18(10). October 2009

Figure 2. Gene copy number alteration as a function of gene expression in 3p12.3-p14.2, 3q26.2-q26.32, and 9p21.3-p23. Accord- ing to array data, genes showing deletion and reduced expression or amplification and overexpression are indicated.

with copy-number loss (copy number variations with ab- number and expression levels, the comparisons of Gain- erration score <0.5 and number of contiguous probe ≥3) TH vs. Up-TH, and Loss-TH vs. Down-TH were made. in NPC-TW01 and HONE1, respectively. Among them, We found that 12.4% (105/850) of Gain-TH genes were 582 were deleted in both cell lines (Loss-TH). The most overexpressed and 15.1% (88/582) of Loss-TH genes were prominent CNVs (copy number variations) observed in- down-regulated. Interestingly, the 3q26.2-q26.32 amplicon cluded gain on 3q26.2-q26.31, and loss on 3p21.2-q12.1, contains 14 genes showing both dosage amplified and 9p21.3-p24.3, and nearly the whole Y chromosome (Sup- overexpressed, and each of the deletion regions 3p12.3- plementary Table S2). Interestingly, three of these four p14.2 and 9p21.3-p23 comprises eight down-regulated prominent CNVs, namely, gain on 3q26.2-q26.31, and loss genes, respectively. The array CGH profiles for chromo- on 3p21.2-q12.1 and 9p21.3-p24.3, have been identified somes 3 and 9 and the altered genes located in the prom- as “hot spots” in a meta-analysis study that used CGH/ inent gain/loss regions are illustrated in Fig. 2. Notably, array CGH data sets obtained from 188 nasopharyngeal the striking deletion region at 3p14.2-p14.3 was not con- carcinoma tumors (27). In an attempt to know the impact sidered a candidate because the expression level of FHIT, of ethnicity, a DNA pool from 10 healthy Taiwanese the only gene involved in this region, did not correlate males was compared with the commercial Caucasian ge- with genomic content. A high-density distribution of al- nome used in this current study. Our results showed that tered expressed genes emphasized the biological signifi- some microdeletions and microamplifications could be cance of changes in copy-number on 3p12.3-p14.2, detected, but no prominent CNVs were found (Supple- 3q26.2-q26.32, and 9p21.3-p23. We also noted that a large mentary Fig. S1), suggesting the defined altered regions number of the altered genes were cancer-related. are related to nasopharyngeal carcinoma development. To validate mRNA expression levels detected by the ex- The genome-wide effect of copy-number aberrations on pression arrays, all of the commonly dysregulated genes transcription was evaluated by parallel conduct of gene mapped to the three prominent copy number alterations expression microarray experiments on NPC-TW01 and were subjected to quantitative real-time PCR. Up to 87.5% HONE1 cells. In order to filter out differential genes un- to 92.9% of genes were cross-validated by both methods, related to carcinogenesis of nasopharyngeal carcinoma, a as summarized in Fig. 3. Six genes were selected as the gene expression database for premalignant nasopharyn- representative genes (two genes for each region) for fur- geal epithelial cells was obtained from NP460hTert cells, ther study because they showed higher fold changes as and we used the commercial universal human RNA compared with the neighboring genes by qPCR valida- (Stratagene) as common reference. As shown in Venn dia- tion. These genes were ADAMTS9 and LRIG1 located at grams in Supplementary Fig. S2A and B, a total of 1,272 3p12.3-p14.2, GPR160 and SKIL at 3q26.2-q26.32, and genes were up-regulated (Up-TH) and 1,810 genes were ADFP and MPDZ at 9p21.3-p23. down-regulated (Down-TH) in both cell lines. To identify Genetic Alterations and Clinical Association. To con- the genes that were altered consistently in DNA copy firm the genetic alterations, dual-color FISH was done to

Cancer Epidemiol Biomarkers Prev 2009;18(10). October 2009

detect the defined genetic altered regions in metastatic (P = 0.0012*; Fig. 5A). A similar finding also was found nasopharyngeal carcinoma tissues in an independent for the 3q26.2-q26.32 amplicon (Fig. 5B). panel of 48 clinical samples. For the 3p12.3-p14.2 deletion Our data indicated that, using the approach of com- region, homozygous deletion (FISH ratio <-2) of bined analyses between genetic alterations and gene ADAMTS9 (red FISH signal)andLRIG1 (green signal) expression profiles, we successfully defined the 3p dele- could be found in 13 (33%) and 12 (30%) of 40 nasopha- tion (3p12.3-p14.2) and 3q amplification (3q26.2-q26.32) ryngeal carcinoma samples respectively (Fig. 4A and B, regions that correlated well with poor patient survival. right). Using the same FISH probe sets, gene loci of Furthermore, our data also support the potential of ADAMTS9 and LRIG1 could be detected in normal or ADAMTS9 and LRIG1 being tumor suppressors and nondeleted nasopharyngeal carcinoma tissues (Supple- GPR160 and SKIL being oncogenes involved in nasopha- mentary Fig. S3A). Small genetic gain of the 3q26.2- ryngeal carcinoma development and progression. Due to q26.32 region (FISH ratios between 1.0 and 2.8) could be the limited number of cases, we did not analyze the clin- foundinmostofthetumortissuesbasedontheFISH ical impact of the 9p21.3-p23 deletion in nasopharyngeal analyses of GPR160 and SKIL. Moderate to high amplifi- carcinoma tumors. cations (FISH ratios >2.8) were verified in 10 of 40 (25%) samples for GPR160 (green signals versus red signals) and in 13 of 40 (33%) samples for SKIL (red versus green) Discussion (Fig. 4A, middle). Similar to the 3p deletion, loss of heterozygosity of ADFP (green versus red) and MPDZ (green ver- This study showed that 3p deletion (3p12.3-p14.2), 3q am- sus red) at 9p21.3-p23 could be found in 6 of 40 (15%) plification (3q26.2-q26.32), and 9p21.3-p23 deletion are nasopharyngeal carcinoma tumors (Fig. 4A, right). FISH common genetic events during nasopharyngeal carcino- analyses on the 3q nonamplified and 9p undeleted ma development. We identified these altered regions in tumors are shown in Supplementary Fig. S3B and C. two well-known nasopharyngeal carcinoma cells, NPC- To determine whether these genetic alterations affect TW01 and HONE1, and later validated the findings the overall survival, Kaplan-Meier survival analysis was by dual-color FISH analysis on a larger scale using done to evaluate the clinical correlation. In this study, paraffin-embedded metastatic nasopharyngeal carcinoma a more stringent criterion was used for subgrouping. tissues (n = 48). The potential tumor suppressor genes, Because there were two candidate genes in each altered ADAMTS9 and LRIG1, and the suspected oncogenes, genetic region, cases with both genes amplified or deleted GPR160 and SKIL, were defined by combined analyses were defined as abnormal and the rest as normal. Based to indicate consistent biological relevance from genomic on this definition, a significant association was found copy alterations to mRNA expression and clinical associ- between 3p12.3-p14.2 deletion and poor clinical outcome ation. This study provides cogent evidence to suggest prognostic potentials of the defined 3p deletion (3p12.3- p14.2) and 3q amplification (3q26.2-q26.32) for clinical application. Nasopharyngeal carcinoma arises from the epithelial cells that cover the surface and line the nasopharynx and typically metastasize to cervical lymph nodes for the advanced tumors. Because nasopharyngeal carcinoma is markedly radiosensitive, radiotherapy is the primary mode of treatment (29, 30). However, there is a high fail- urerateforsuchtreatmentoncethetumorspreadsto other organs and becomes metastatic (31). Identification of prognostic indicators that correlate with the clinical outcome would be helpful to develop new treatments for the severe types of nasopharyngeal carcinoma. For such clinical limitation, we focused particularly in this study on identifying novel genetic markers in metastatic tumors. The biological roles of newly defined candidate genes during nasopharyngeal carcinoma development are under investigation. Gene amplification accounts for genomic instabilities in solid tumors (32). It has been proposed that activation of proto-oncogenes by amplification plays an important role in the development of many human cancers. Therefore, detection of specific gene amplifications in cancer cells can lead to the identification of putative oncogenes involved in growth control and metastasis. Our results con- firm the previous conclusion that 3q26 is the principal amplified region in nasopharyngeal carcinoma genomes Figure 3. Microarray and quantitative real-time PCR cross- (17, 22-24, 33). Taking advantage of high-resolution array validated the changes of expression level of genes located on platforms, we have narrowed down the amplified region 3q26.2-q26.32 amplicon and 3p12.3-p14.2 and 9p21.3-p23 to 3q26.2-q26.31 (around 3.5 Mb) in two nasopharyngeal deletion regions. Stars, genes significantly up-regulated or carcinoma cell lines and pinpointed several target genes down-regulated and chosen for further study. within these defined regions. Among these genes, 13 also

Cancer Epidemiol Biomarkers Prev 2009;18(10). October 2009

Figure 4. FISH analysis on nasopharyngeal carcinoma tissue microarray. A. The distribution of FISH signal ratio of nasopharyngeal carcinoma tissue samples and the detection rate is indicated under each FISH probe. The definition of FISH signal ratio for gene amplification or deletion is described in Materials and Methods. B. The representative FISH images for loss of ADAMTS9 (target gene versus control gene, red signal versus green signal), LRIG1 (green versus red), ADFP (green versus red), and MDPZ (green versus red), and amplification of GPR160 (green versus red) and SKIL (red versus green).

have been found overexpressed by gene expression mi- study provides the first evidence of involvement of croarray and qPCR experiments. Consistent with these re- GPR160 in nasopharyngeal carcinoma development sults, FISH analysis on TMA slides for SKIL (SKI-like and progression. oncogenes, 3q26) and GPR160 (G protein-coupled recep- Of note, the prominent amplification region 3q26.2- tor 160, 3q26.2-q27) revealed a high incidence of amplifi- q26.31 detected in this study is precisely coincident with cation of these two genes in malignant nasopharyngeal one of the amplification regions observed in naso- carcinoma tumors. In particular, the occurrence of ampli- pharyngeal carcinoma cells harboring recurrent EBV reac- fication of SKIL was up to 33% of the samples tested. tivations (12). This finding not only strengthens the SKIL, also known as SnoN, is a member of the Ski family involvement of EBV in the pathogenesis of nasopharyn- of proto-oncogenes. Elevated SKIL expression has been geal carcinoma, but also provides supporting evidence observed in many human cancer types, including lung, for our hypothesis that frequent EBV reactivation may en- stomach, breast, ovary, and thyroid (34-36). The role of hance genome instability, in nonrandomly distribution SKIL in tumorigenesis is important but complex. Using patterns, and accelerate the progression of nasopharyn- human lung cancer and breast cancer cell lines, Zhu geal carcinoma (11, 12). On the other hand, these results et al. showed that SKIL promotes mitogenic transforma- suggest that 3q26.2-q26.31 also may be a critical region for tion at the early stages of tumorigenesis while inhibiting nasopharyngeal carcinoma malignancy, and therefore epithelial-to-mesenchymal transdifferentiation at later overexpressed genes located in this region warrant fur- stages of tumor development (37). Further study is needed ther detailed analysis. to reveal the roles SKIL plays in nasopharyngeal carcino- Loss of 3p and 9p are other genomic abnormalities that ma and the functions of other genes mapped onto the 3q have been described frequently (13, 15, 41-44). Especially amplification region. GPR160 was defined as a member of for 3p, this is the most commonly deleted region in naso- the G protein-coupled receptor family (38, 39). Although pharyngeal carcinoma, as already known. Moreover, loss known to be a signaling molecule, the binding ligands of 3p has been identified as an early event in the tumor- and cellular functions of this gene in cancer develop- igenesis of nasopharyngeal carcinoma (21). Again, we ment are largely unknown. Up-regulation of GPR160 have refined the affected regions to 3p12.3-p14.2 and has only been reported in prostate cancer (40) and our 9p21.3-p23, as well as pinpointed potential tumor

Cancer Epidemiol Biomarkers Prev 2009;18(10). October 2009

ified as important genes involved in EBV reactivation (11). Interaction or combination between different genes within the defined regions may provide survival advantages for cancer development/progression, and the molecular mechanism needs further investigation.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Acknowledgments The costs of publication of this article were defrayed in part Figure 5. Kaplan-Meier survival analysis for nasopharyngeal by the payment of page charges. This article must therefore carcinoma patients. The overall survival for patients was com- be hereby marked advertisement in accordance with 18 U.S.C. pared based on FISH signal ratios of the defined genetic altered Section 1734 solely to indicate this fact. regions: A. 3p12.3-p14.2. B. 3q26.2-q26.32. We thank Dr. Chyi-Chyang Lin (China Medical University Hospital, Taiwan) for his invaluable comments on this article and Ms. Yu-Wen Chiu (National Health Research Institutes, suppressor genes within these deleted regions by coupled Taiwan) for her technical support. We are grateful to Dr. Tim analysis profiles of array CGH and gene expression array. J. Harrison of UCL Medical School, London, UK, for critical There is a high density of down-regulated genes located reading of the manuscript. in the deleted 3p12.3-p14.2 and significant association of this genomic deletion with lower survival rates. These References findings highlight the importance of genes detected in 1. Muir CS. Nasopharyngeal carcinoma in non-Chinese populations 3p12.3-p14.2. with special reference to south-east Asia and Africa. Int J Cancer Among the genetic markers described here, both 1971;8:351–63. ADAMTS9 and LRIG have been reported to be putative 2. Simons MJ, Wee GB, Day NE, Morris PJ, Shanmugaratnam K, De-Thé GB. Immunogenetic aspects of nasopharyngeal carcinoma: I. Differ- tumor suppressor genes during cancer development ences in HL-A antigen profiles between patients and control groups. (45-47). It is of particular note that gene silencing of Int J Cancer 1974;13:122–34. ADAMTS9, mapping to 3p14.2-p14.3, was recently found 3. Chan AS, To KF, Lo KW, et al. 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Jim Jinn-Chyuan Sheu, Chia-Huei Lee, Jenq-Yuh Ko, et al.

Cancer Epidemiol Biomarkers Prev 2009;18:2709-2716.

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