Genomic Alteration in Head and Neck Squamous Cell Carcinoma (HNSCC) Cell Lines Inferred from Karyotyping, Molecular Cytogenetics, and Array Comparative Genomic Hybridization

Genomic alteration in head and neck squamous cell carcinoma (HNSCC) was studied in two cell line pairs (HN30-HN31 and HN4-HN12) using conventional C-banding, multiplex fluorescence in situ hybridization (M-FISH), and array comparative genomic hybridization (array CGH). HN30 and HN4 were derived from primary lesions in the pharynx and base of tongue, respectively, and HN31 and HN12 were derived from lymph-node metastatic lesions belonging to the same patients. Gain of chromosome 1, 7, and 11 were shared in almost all cell lines. Hierarchical clustering revealed that HN31 was closely related to HN4, which shared eight chromosome alteration cases. Large C-positive heterochromatins were found in the centromeric region of chromosome 9 in HN31 and HN4, which suggests complex structural amplification of the repetitive sequence. Array CGH revealed amplification of 7p22.3p11.2, 8q11.23q12.1, and 14q32.33 in all cell lines involved with tumorigenesis and inflammation genes. The amplification of 2p21 (SIX3), 11p15.5 (H19), and 11q21q22.3 (MAML2, PGR, TRPC6, and MMP family) regions, and deletion of 9p23 (PTPRD) and 16q23.1 (WWOX) regions were identified in HN31 and HN12. Interestingly, partial loss of PTPRD (9p23) and WWOX (16q23.1) genes was identified in HN31 and HN12, and the level of gene expression tended to be the down-regulation of PTPRD, with no detectable expression of the WWOX gene. This suggests that the scarcity of PTPRD and WWOX genes might have played an important role in progression of HNSCC, and could be considered as a target for cancer therapy or a biomarker in molecular pathology.


Introduction
Genomic reorganizations have played an important role in the process of tumor development from a single precursor cell to invasive carcinoma. The occurrence of non-homologous recombination and gene conversion result in chromosomal rearrangements (translocations, insertions, or deletions), amplifications, point mutations, and epigenetics, which often alter the function of proteins [1,2]. A consequence of chromosome number alteration and genomic copy number variations (CNVs) is the dysregulation of proto-oncogenes or tumor suppressor gene expression, leading to numerous types of dysplasia and neoplasia [3].
Head and neck squamous cell carcinoma (HNSCC) is one of the major causes of global cancer-related mortality, estimated at between 223,000 and 300,000 deaths per year [4]. From 2002 to 2004, 1,186 head and neck cancer cases were diagnosed in Thailand, consisting of 34.6% oral cavity cases, 30.1% oropharynx cases, 16.7% hypopharynx cases, and 18.6% larynx cases [5]. Major risk factors are known to be tobacco use, alcohol consumption, betel quid chewing, and bidi smoking. Over 26,000 Thai people were diagnosed with head and neck cancers in 2010 [6]. Although numerous advances in diagnosis and treatment of oral cancer are available, mortality and morbidity rates for head and neck cancers are still high. This could reflect a high variation of genetic instability or molecular heterogeneity, and complexities of subcellular abnormalities through oral carcinogenesis. Several reports have investigated the molecular mechanisms of HNSCC development [3,7,8]. However, most analyzed gene expression patterns with small populations, and tumor progression stages from different patients. It is necessary to confirm the results from larger groups of patients, because HNSCCs are probably derived from different subsites within the oral cavity, known as 'field cancerization' [9]. Within a single tumor mass, the occurrence of a single clone that evolves into different subpopulations, such as 'second field tumor' (SFT), resulting from accumulation of diverse genetic variation, is designated as the clonal evolution model [10]. Two genetically heterogeneous subpopulations coexist within a single tumor mass, and the predominant population is probably replaced by other subpopulations based on the effects of environmental selection pressure and/ or the stage of tumor progression from primary tumor to lymph node metastasis [11]. Alternatively, if the individual tumors show different genomic alterations within the same site, the secondary lesion might be regarded as a second primary tumor (SPT) [12]. The study of genome profiling of HNSCC is required to elucidate genetic loci relating to tumor classification and homogeneity, novel diagnosis, and therapeutic clinical management. Previous research has elucidated the genomic profile of HNSCC or cell lines using comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification (MLPA) to determine various CNVs [13,14]. However, these methods were not able to detect balanced chromosomal rearrangements (e.g., translocations or inversions), interchromosomal rearrangements, and low frequency mosaicism. Cytogenetic techniques such as karyotyping and multiplex fluorescence in situ hybridization (M-FISH) are thus needed to extensively examine genome alterations in HNSCC.
The study of tumor cell lines is a useful strategy to obtain insights into cell-specific gene regulation. Many tumor cell lines have been studied to identify regions which relate to oncogenic or tumor-suppressive genes in HNSCC [3]; however, little data is available on primary tumor and metastasis derived cell lines isolated from the same patients [15,16]. The relevance of chromosome alteration and genomic CNVs, corresponding to the stage of phenotypic progression and invasive malignancy, has not been fully characterized. The HN cell lines are commonly used for cellular function studies. Transcriptomic and proteomic analyses in HNSCC have been obtained from these cell lines [3,13,15,17]. In this study, the genome profiling of four HN cell lines (HN30, HN31, HN4, and HN12) derived from two patients was analyzed. Two cell lines were primary tumor, and the other two were lymph node metastases. We performed multiplex-fluorescence in situ hybridization (M-FISH) to investigate structural and numerical chromosome alterations, and analyzed cell clonality within each cell line by hierarchical clustering analysis. The heterochromatin distribution was also examined using C-banding in each cell line. High-resolution array comparative genomic hybridization (array CGH) was then conducted to identify CNVs. The mRNA expression of specific genes was identified using real time quantitative reverse transcription PCR (real time qRT-PCR), and the candidate genes and regions that related to the molecular pathology and tumor progression in HNSCC were discussed.

Cell lines
Head and neck squamous cell carcinoma (HNSCC) cell lines (HN30, HN31, HN4, and HN12) were provided by J. Silvio Gutkind (NIDCR, Bethesda, MD, USA). HN30 (T3N0M0) and HN31 (T3N0M0) were both derived from the same male patient, and HN4 (T4N1M0) and HN12 (T4N1M0) were derived from another male patient. HN30 and HN4 were obtained from primary lesions in the pharynx and the base of tongue, respectively. HN12 and HN31 were derived from lymph node metastases [17]. Whole genomic DNA was isolated from the four HNSCC cell lines using a salting out method as described previously [18]. Cross-contamination for each cell line was examined using STR DNA profiling analysis (Investigator1 IDplex Plus, Qiagen, GmbH, Hilden, Germany) to confirm the purity of each cell line (data not shown). A human mucoepidermoid pulmonary carcinoma NCI-H292 (ATCC number CRL-1848), and a non-tumorigenic human skin keratinocyte cell line HaCaT provided by Stitya Sirisingha (Mahidol University, Thailand) were used as control groups in the expression analysis of candidate genes. This study was approved by the Institute Biosafety Committee of Thammasart University (TU-IBC, Approval No 011/2558).
Cell culture and chromosome preparation HN30, HN31, HN4, and HN12 were cultured in Dulbecco's modified Eagle's medium (Life Technologies-Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies-Gibco), 100 mg/ml penicillin-streptomycin (Life Technologies-Gibco), 100 mg/ ml Amphotericin B and incubated at 37°C in a humidified atmosphere of 5% CO 2 in air. Chromosome preparation was conducted as described previously [19]. Briefly, before harvest, HNSCC cells in the logarithmic growth phase were incubated with 120 ng/ml colcemid (Life Technologies-Gibco) for 45 min. The cells were harvested by treatment with 0.025% trypsin-EDTA, suspended in 0.075 M KCl at room temperature for 20 min, and fixed with methanol/ acetic acid (3:1) three times. The cell suspension was dropped on cleaned glass slides and airdried. The slides were kept at -80°C until used. HaCaT and NCI cell lines were cultured in medium under conditions described previously [20,21].
(Sigma-Aldrich) as described previously [23]. The labeled products were pooled and sonicated to achieve a size range of 200-1,000 base pairs, optimal for use in chromosome painting. Then, the sonicated DNA samples were precipitated with ethanol together with human Cot-1 DNA (Thermo Fisher Scientific, Waltham, MA USA) and resuspended in a hybridization buffer (50% formamide, 2 × SSC [saline sodium citrate], 10% dextran sulfate, 0.5 M phosphate buffer, 1 × Denhardt's solution [pH 7.4]). Metaphase spreads on slides were denatured by immersing in an alkaline denaturation solution (0.5 M NaOH and 1.0 M NaCl) for 50-60 s, followed by rinsing in 1M Tris-HCl (pH 7.4) solution for 3 min, 1 × PBS for 3 min, and dehydration through a 70, 90, and 100% ethanol series. The M-FISH probe was denatured at 65°C for 10 min before being applied onto the denatured slides. The hybridization area was sealed with a 22 × 22-mm coverslip and rubber cement. Hybridization was carried out in a 37°C incubator for 2 nights. The posthybridization washes included a 5-min stringent wash in 0.5 × SSC at 75°C, followed by a 5-min rinse in 2 × SSC containing 0.05% Tween20 (VWR, Radnor, PA, USA) and a 2-min rinse in 1 × PBS, both at room temperature. Finally, the slides were mounted with SlowFade Gold mounting solution containing 4 0 6-diamidino-2-phenylindole (Life Science, Waltham, MA, USA). Images were visualized on a Zeiss AxioImager D1 fluorescent microscope equipped with narrow band-pass filters for DAPI, DEAC, FITC, Cy3, Texas RED, and Cy5 fluorescence and an ORCA-EA CCD camera (Hamamatsu, Shizuoka, Japan). M-FISH digital images were captured using the SmartCapture software (Digital Scientific, Cambridge, UK) and processed using the SmartType Karyotyper software (Digital Scientific). Ten metaphase cells per cell line were karyotyped.

C-banding
To examine the chromosomal distribution of constitutive heterochromatin, C-banding was performed using the standard barium hydroxide/saline/Giemsa method [24] with the following slight modification: chromosome slides were treated with 0.2 N HCl at room temperature for 60 min, and then with 5% Ba(OH) 2 at 50°C for 1 min, followed by 2× SSC at 65°C for 60 min.

Hierarchical clustering
A cluster analysis was performed to assess the chromosome alterations among the four cell lines by considering the type of chromosome aberrations within metaphases. Each aberration that was recorded in over five of the ten metaphase cells was computed as present or absent within the karyotype of different metaphases. The frequency (%) of each chromosome alteration was then compared among the four cell lines by Cluster 3.0 software [25] using the commonly used average linkage method and visualized with Java TreeView 1.1.6 [26].

Array comparative genomic hybridization (array CGH)
Whole genomic DNA was used as a template for array CGH analysis with 60-mer oligonucleotide CGH arrays (Agilent Technologies, Santa Clara, CA, USA). Briefly, 20 μg of genomic DNA in each cell line and reference genomic DNA [Hg19; Genome Reference Consortium Human Reference (GRCh37), http://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.13/] were digested using AluI and RsaI (Agilent Technologies). The digested tumor and reference DNAs were labeled with cyanine 3-dUTP (Cy3-dUTP) and cyanine 5-dUTP (Cy5-dUTP), respectively, using SureTag DNA Labeling Kit (Agilent Technologies) following the manufacturer's instruction. Equal amounts of tumor and reference DNAs were subsequently pooled and mixed with human Cot-1 DNA (1.0 mg/ml), dissolved in Hybridization Master Mix, 10× a CGH Blocking Agent and 2× HI-RPM Hybridization Buffer (Agilent Technologies), denatured and hybridized to the CGH array at 65°C for 24 h. Glass slides were washed and then scanned in accordance with the manufacturer's instructions. Microarray images were analyzed using Agilent C microarray scanner (Agilent Technologies) with linear normalization according to manufacturer's instructions, and the data were imported into Default Analysis Method-CGH v2 (Agilent Technologies). After normalization of the raw data, the log2 ratio of Cy3 (tumor) to Cy5 (reference) was calculated. Aberrant regions were determined using the Aberration Detection Method-2 (ADM-2) algorithm at a threshold of 6.0. To detect amplification and deletion, we set the values of parameters for the aberration filters as: minimum number of probes for amplification ! 3, nesting level 100, minimum average, absolute log ratio for amplification ! 0.25, minimum size (Kb) of region for amplification ! 0.0, minimum size (Kb) of region for deletion ! 0.0, minimum number of probes for deletion ! 3, and minimum average, absolute log ratio for deletion ! 0.25. Differences at P 0.05 were considered to be statistically significant.

RNA isolation and expression analysis of candidate genes
The levels of four gene [SIX3 (2p21), H19 (11p15.5), PTPRD (9p23), and WWOX (16q23.1)] expressions were detected from four HNSCC cell lines, and control group: HaCaT and NCI. All cell lines were lysed with TRIzol Reagent (Life Technologies-Invitrogen, Carlsbad, CA, USA), and total RNA was isolated following the manufacturer's instructions. cDNA fragments were synthesized using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, California USA), and used as real time PCR templates to amplify the four gene fragments. The mRNA expression of these four genes was measured by Bio-Rad iCycler Thermal Cycler with iQ5 Multicolor Real Time PCR Detection System (Bio-Rad) as signal reporter. Fifty nanograms of cDNA amplification was carried out in 10 μl of Master Mix that contained 2X iTaq TM Universal SYBR 1 Green supermix (Bio-Rad), and 10.0 μM gene-specific primers (Table 1), designed from the NCBI reference sequence of SIX3 (LC106310), H19 (LC106311), PTPRD (LC106313), and WWOX (LC106312) genes. PCR conditions were as follows: an initial denaturation at 95°C for 30 s, followed by 50 cycles that each involved incubation at 95°C for 15 s, 58°C for 30 s and 95°C for 1 min, and 55°C for 1 min. Dissociation melting curves, created by running a heat dissociation protocol after the PCR (81 cycles of 55.0°C-95.0°C for 10 s, increase set point temperature by 0.5°C), confirmed the specific amplification of cDNA target and the absence of nonspecific products. The GAPDH gene was also used as the internal reference to normalize the expression levels between samples, using GAPDH_01_F (5 0 -GATAACGGATTTGGTCGTATTG-3 0 ) and GAPDH_01_R (5 0 -CATGGGTGGAATCATATTGGAA-3 0 ) primers [27]. Quantitative real time PCR reactions of all cell lines were performed in biological triplicates. Threshold cycle (Ct) values were used to calculate ΔCt values from the products of GAPDH and SIX3, H19, PTPRD, and WWOX genes for all cell lines. The relative expression levels of different cell lines were calculated using the 2 −ΔΔCt method [28]. Statistical differences of SIX3, H19, PTPRD, and WWOX genes between cell lines were detected by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test, using GraphPad Prism V5 (GraphPad Software, La Jolla, CA, USA), and the level of statistical significance was tested and represented as Ã for P 0.05, ÃÃ for P 0.01, and ÃÃÃ for P 0.001. Estimated values were expressed as mean ± standard deviation. The PCR products of cDNA fragments from SIX3, H19, PTPRD, and WWOX genes were also cloned using pGEM-T Easy Vector System I (Promega, Madison, WI, USA), and the nucleotide sequences of the DNA fragments were determined using 1 st

Discussion
Genomic alteration of four HNSCC cell lines was characterized by M-FISH and array CGH. Chromosome alteration profiles were analyzed for each cell line, and the types and frequencies of abnormalities were observed using hierarchical clustering. No shared structural aberration case was found in HN30 and HN31, HN4 and HN12, or in primary lesion (HN30 and HN4) and metastatic lesion (HN31 and HN12). However, most of the cell lines shared numerical aberrations with gain of chromosome 1, 7, and 11. Basically, both chromosome 7 and 11 contain genes that are involved in the tumor progression such as EGFR, HGF, HOX, BRAF, PAFAH1B2, FLI1, and ETS1 [30][31][32][33][34][35], in particular LAMB1, MET, MMP1, CCND1, ORAOV1, FADD, PPFIA1, and CTTN genes which related to HNSCC [36][37][38]. In addition, the gain of chromosome 7 was predominantly found in colorectal cancer, glioblastoma, and renal cell carcinoma [32,39,40], and the gain of chromosome 11 was reported in ovarian cancer and myeloid leukemia [41,42]. This suggests that the gain of both chromosome 7 and 11 have significance in carcinogenesis, when compared to other chromosomes in numerical aberration. Further investigations are required to elucidate the relationship between chromosome 7 and 11. Most numerical and structural aberrations were often found in chromosome 1, which related to genes (COL11A1, GSTM3, CKS1B, CDT2, ETV3, ELF3, ASPM, KIF14, NEK2, DTL, CENPF, CKS1B, EXO1, GPR137B, and CD1D) in carcinogenesis [43][44][45][46][47][48]. No shared chromosome number aberration case was found in HN30 and HN31, or in primary lesion (HN30 and HN4) and metastatic lesion (HN31 and HN12), except for the loss of chromosome 4 found in HN4 and HN12. This chromosome contains genes that are involved in carcinogenesis such as PDLIM3, ANK2, SORBS2, and PPARGCIA [49]. Hierarchical clustering analysis revealed several clusters in each cell line, suggesting that tumor heterogeneity was found in both primary and metastatic lesions. Surprisingly, the consequence of clustering with four cell lines suggested that three major clusters were grouped as HN30, HN12, and HN31-HN4. HN31 was closely related to HN4, which shared eight chromosome alteration cases, most of which were structural aberrations. This suggests the possibility of complex structural aberration groupings of HN31 and HN4 into the same cluster analysis. Although this cluster relationship was not correlated to the tumor clinical stages, previous studies showed the same phenotypic progress with the low level of MMP9 (20q13.12) activity in the potential of HN31 and HN4 invasion, but not for HN30 and HN12 [16,50]. Moreover, large C-positive heterochromatins were found in the centromeric region of chromosome 9 for both HN31 and HN4, which suggests the specific amplification of repetitive sequences in these two cell lines. However, the results of array CGH showed many different CNVs between the two cell lines (HN31 and HN4). The grouping of the two cell lines might result from analyzing a few markers (chromosome alteration types) that make close relationship unintentional.
On the basis of chromosome alterations in this study, the presence of tumor progression from a single precursor cell might be explained by the occurrence of aneuploidy, polyploidy, and deletion, followed by various chromosome structural aberrations such as translocations and insertions [51][52][53]. This agreed with similar numbers of numerical aberration among four cell lines (HN30 = 25 cases, HN31 = 26 cases, HN4 = 22 cases, and HN12 = 24 cases). By contrast, the number of structural aberration cases tended to occur in HN12 (58 cases), which is the metastatic cell line in stage four of cancer. This suggests that structural alterations were predominantly observed in invasive malignancy [54].
Genome wide profiling of the four cancer cell lines was alternatively carried out using high resolution array CGH, at a resolution of approximately 0.06 kb with 180,000 probes to determine CNVs. Large CNVs were found in 5p15.33p11 (amplification), 7p22.3p11.2 (amplification), 8q11.23q24.3 (amplification), which were also reported as chromosome arm level CNVs in the previous work [55]. We were able to identify large additional regions: 9q13q34.3 (amplification), 10p15.3p11.1 (deletion), 14q11.2q32.33 (amplification), 20p13p11.1 (amplification), and 20q11.  [56][57][58][59][60][61], all of which were involved in tumor cell proliferation and angiogenesis in oral carcinogenesis [50,[62][63][64][65]. Notably, the most complex patterns of amplifications or deletions were identified as 22q11.21 region in HN30, HN31, and HN12, and this region comprises TBX1 that also involved negative regulator of tumor cell growth [66]. Another site was observed in the 4q13.2 region of HN31 and HN12, and found both amplification and deletion in HN30 in this study. This phenomenon was also observed in studies by Ambatipudi et al. [61] and Vincent-Chong et al. [55] which suggests possible tumor heterogeneity. The 4q13.2 region contained the UGT2B17 gene that involved tongue and larynx SCC. The copy number polymorphisms of this gene in cancer are required to understand the role of this gene in HNSCC.
Three common regions of amplification of chromosome segments found in all four cell lines were related to PDGFA, MAD1L1, ACTB, IL6, CYCS, GLI3, DDC, EGFR, SDK1, KIAA0125, and ADAM6 genes. These genes involved tumorigenesis and inflammation in various tumor types [30,[67][68][69][70][71][72][73][74][75][76][77]. Three regions: 3p14.2 (deletion), 9p21.2q21.31 (amplification), and 20q13.12 (amplification) were commonly found in HN4 and HN31 as the same cluster in our analysis, and five genes (FHIT, MMP9, GNA14, GNAQ, and PSAT1) were involved in tumorigenesis in lung cancer and colon cancer [50,63,[78][79][80]. One region: 1p13.2 (deletion) was found in both HN30 and HN4, derived from primary lesion stages, and SYCP1 gene involved in carcinogenesis in medulloblastoma [81]. Five regions: 2p21 (amplification), 11p15.5 (amplification), 11q21q22.3 (amplification), 9p23 (deletion), and 16q23.1 (deletion) were commonly found in HN31 and HN12. These genes: SIX3, H19, PTPRD, WWOX, MAML2, PGR, TRPC6, MMP20, MMP13, MMP1, and MMP3 were involved in tumor progression of mucoepidermoid carcinoma, prostate adenocarcinoma, glioblastoma, pancreatic cancer, lung cancer, and epithelial breast cancer [50,[82][83][84][85][86][87][88][89][90]. To extensively investigate the regulation of these functional genes, four of five regions: 2p21 (SIX3), 11p15.5 (H19), 9p23 (PTPRD), and 16q23.1 (WWOX) that showed amplification or deletion in both HN31 and HN12 were examined for the level of gene expression by qRT-PCR. Sine oculis homeobox homolog 3 (SIX3) gene plays a role in embryogenesis in vertebrates, as a transcription repressor to regulate proliferation and metastasis related genes [89]. Basically, genomic amplification which increases gene dosage can lead to over-expression at mRNA level. This agreed with the level of SIX3 expression in HN12, but not for HN31 whose SIX3 expression was the same level with the control group (HaCaT and NCI). Over-expression was also found in HN30, though no amplification or deletion were found in array CGH. This suggests that the number of SIX3 gene copy is not related to gene expression in HNSCC. The mechanism of SIX3 over-expression remains unclear, and further upstream analysis is required to elucidate its role in HNSCC. A similar case was found in H19, which is an imprinted oncofetal gene with maternal expression in fetal tissues. It is expressed in the first stage of embryogenesis, but down-regulated or turned off in most tissues after birth as a tumor suppressor role [84,91]. Studies of various tumors have demonstrated an overexpression of the H19 RNA when compared to normal tissues, leading to targeting for gene therapy [92,93]. However, both HN31 and HN12 showed very low levels of expression for H19. This pattern was also found in HN4 and NCI, whereas HN30 showed the same expression level as found in HaCaT. Human bladder carcinoma derived from adult bladder mucosa cells had also lost their ability to express H19 gene. However, the significant increase of H19 during the process of tumor formation in mice was accomplished by injected carcinoma cell lines [93]. This suggests that H19 is expressed at substantial levels in several different tumor types [94], and might re-activate expression within the process of tumorigenesis. Protein tyrosine phosphatase receptor type D (PTPRD) is a receptor type tyrosine-protein phosphatase (membrane-integral enzymes) that helps to remove phosphate groups from specific proteins [95]. Mutation or epigenetic silencing of PTPRD has been reported for the loss of tumor suppressor role in several cancers [96,97]. In this study, although trisomy was found on chromosome 9 in HN31 and HN12, the presence of deletion still occurred at PTPRD. This agreed with the loci of genomic deletion which reduced gene dosage and resulted in down-expression at mRNA level in metastatic cells (HN31 and HN12) compared to less invasive cells (HN30 and HN4), suggesting that the loss of PTPRD function involved cell migration [98].
Chromosome region 16q23.3q24.1 is the same locus with the fragile site (FRA16D) and often breaks down in several cancer types [82,99]. WW domain containing oxidoreductase (WWOX) gene, which is also located in these regions is considered an important role in tumor suppression through transcriptional repression and apoptosis [100]. Mutation and epigenetic silencing of the WWOX gene have been reported in various cancers [101][102][103], and the loss of WWOX expression is an important step in carcinogenesis [101,104]. Although deletions of 16q23.1 were not found in HN30 and HN4, low levels of WWOX expression were observed in all cell lines. This agreed with many tumor types affected by WWOX deletions [102], which suggests that the loss of WWOX at both genomic or expression level is an important role of cell development in carcinogenesis. This gene is also a promising target to develop gene therapy plan [100,105].
In this study, the genome profiles of primary tumor and metastasis derived cell lines isolated from the same patients were addressed by karyotyping, molecular cytogenetics, and array CGH. Results suggest that segments/regions corresponded to the stage of tumor progression/ carcinogenesis. The presence of CNVs are further needed to gain insight into the association of clinical outcomes in HNSCC, compared to other cancers. Although the level of gene expression is not significantly different between the primary and metastatic pairs for any of the four genes, it is also interesting to understand the role of PTPRD in invasive processes and WWOX genes in carcinogenesis, or as biomarkers to reflect molecular progression in pathogenesis. Transcriptomics and proteomics analysis for these four cell lines are required to gain insight into validating relevant targets. Many candidate genes from the present genomic alteration data are also required to further examine the precision, incidence, and significant target in HNSCC.