Oral Oncology (2007) 43, 508–514

ava i lab le at www.sc iencedi rec t . com

journal homepage: ht tp : / / in t l .e lsevierheal th .com/ journals /oron/

Amplifications of TAOS1 and EMS1 genes inoral carcinogenesis: associationwith clinicopathological features

Juan Xia a,b, Qianming Chen a,*, Bingqi Li a, Xin Zeng a

a Department of Oral Medicine, West China College of Stomatology, Sichuan University, No. 14, Sec. 3,Renminnan Road, Chengdu, Sichuan 610041, People’s Republic of Chinab Department of Oral Medicine, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong510055, People’s Republic of China

Received 2 April 2006; received in revised form 8 May 2006; accepted 9 May 2006Available online 26 September 2006

Summary Amplification of chromosomal region 11q13 is one of the genetic alterations mostfrequently observed in oral squamous cell carcinoma (OSCC). Both TAOS1, a recently identifiedgene, and EMS1 were thought as two important target oncogenes for driving 11q13 amplifica-tion, and their contributions to oral carcinogenesis were hypothesized. Therefore we investi-gated amplifications of TAOS1 and EMS1 genes and their relations to clinicopathologicalvariables in premalignant lesions (leukoplakias) and primary OSCC. TAOS1 amplification, begin-ning from mild-dysplastic epithelia, occurred in 33.3% of leukoplakias and 51.5% of OSCC. EMS1amplification, beginning from moderate-dysplastic epithelia, occurred in 20% of leukoplakiasand 57.6% of OSCC. Both gene amplifications were significantly related to different stages oforal carcinogenesis (p < 0.05). During multistage carcinogenesis, no gene amplification wasobserved in normal tissue and non-dysplastic leukoplakias while, in OSCC with metastasis,amplification frequency increased significantly (p < 0.005). Both TAOS1 and EMS1 amplificationswere significantly associated with larger tumor size, presence of lymph node metastasis, poorhistological differentiation and advanced clinical stage. Our data suggested potential roles inoral carcinogenesis and that TAOS1 might be involved earlier than EMS1. Both genes mightbe candidate biomarkers for diagnosis and prognosis in OSCC.

�c 2006 Elsevier Ltd. All rights reserved.

KEYWORDSTAOS1;EMS1;Oral;Premalignant lesion;Squamous cellcarcinoma

13do

orhe*

68-8375/$ - see front matter �c 2006 Elsevier Ltd. All rights reserved.i:10.1016/j.oraloncology.2006.05.008

Abbreviations: TAOS1, tumor amplified and overexpressed seqence 1; EMS1, ems1 sequence; ORAOV1, oral cancer overexpressed 1; OPL,al premalignant lesion; OLK, oral leukoplakia; OSCC, oral squamous cell carcinoma; HNSCC, head and neck squamous cell carcinoma; H&E,matoxylin & eosin.Corresponding author. Tel.: +86 28 85405251/13880535268; fax: +86 28 85405251.E-mail address: ianchen63@vip.sina.com (Q. Chen).

Table 1 Clinical characteristics of the studied populations(n = 78)

Characteristics Control(n = 15)

OLK(n = 30)

OSCC(n = 33)

Age (yrs)Mean ± SD 53.3 ± 5.3 57.7 ± 6.5 60.3 ± 12.4Range 40–59 40–70 36–83

GenderMale 8 14 24Female 7 16 9

SiteTongue 4 19 17Buccal 1 6 6Gingiva 3 2 6Palate 0 2 3Oral floor 0 1 1Lip 7 0 0

Amplifications of TAOS1 and EMS1 genes in oral carcinogenesis 509

Introduction

Gene amplification, one of the major mechanisms for acti-vation of oncogenes, leads to an increase of DNA copy num-bers and overexpression of oncogenes in many humantumors, contributing to the growth advantage of cells,which subsequently changes their biological behaviors andcauses carcinogenesis.1–3 Chromosomal band 11q13 is a fre-quently amplified genomic segment in a large number of hu-man tumors and is thought as a potential biomarker fordiagnosis and prognosis.4,5 Many oncogenes related to oralsquamous cell carcinoma (OSCC) reside in the amplified11q13 and each gene individually may confer different prop-erties to cancers. TAOS1 was a recently identified gene,which was amplified and overexpressed in 63% of OSCC celllines.6 Accumulated evidence indicates that in addition toCCND1, TAOS1 and EMS1 are the other two important targetoncogenes for driving the 11q13 amplification in OSCC.6 Theaberrant regulation of EMS1 gene contributes to tumor cellinvasion and metastasis by organizing the cytoskeleton andcell adhesion structures.7,8 The dysregulation of cell cycleand cell proliferation due to CCND1 amplification might playan important role in oral carcinogenesis.9,10 Given the closeproximity of TAOS1 to CCND1 (approximately 11.1 kb telo-meric to CCND1 gene) and its high amplification frequencyin OSCC cell lines, it could be interesting to hypothesizeits cancer-related functions. As yet, until now, the naturesand roles of TAOS1 and EMS1 genes in oral premalignant le-sion (OPL) and primary OSCC are still let to be known.

Leukoplakic oral epithelia are a useful model for moni-toring genetic abnormalities and exploring oral carcinogen-esis at cellular level. In this study, we set out to investigateTAOS1 and EMS1 amplifications in surgically resected pri-mary OSCC and oral leukoplakia (OLK) specimens with vari-ous degrees of epithelial dysplasia. In addition, we tried toexamine their clinical and prognostic relevance.

Materials and methods

Subjects and samples

A total of 78 subjects, including 30 OLK patients, 33 OSCCpatients and 15 healthy persons, were enrolled in the pres-ent study from West China Hospital of Stomatology, SichuanUniversity, China. The subjects were aged between 36 and83 years (mean ± SD; 57.9 ± 9.5) and they consisted of 46males and 32 females. Table 1 presents the characteristicsof enrolled subjects. 13 OSCC patients accompanied regio-nal lymph node metastasis, and the remaining (n = 20) didnot. No subjects had radiotherapy, chemotherapy or otherinterventional palliative or therapeutic measures prior tosampling. All subjects participated in this study with writtenconsents and the project was approved by the InstitutionalReview Board.

All surgically resected specimens were formalin-fixedand paraffin-embedded using conventional histopatholo-gical techniques. Histopathological evaluation was per-formed according to the WHO criteria for histologicaltyping of cancer and pre-cancer of the oral mucosa bythe Department of Pathology, West China Hospital of Sto-

matology. Epithelial dysplasia was graded as none (hyper-plasia, n = 6), mild (n = 9), moderate (n = 8), and severe(n = 7).

Microdissection and DNA extraction

Sequential 36 tissue sections (10 lm) stained with hematox-ylin were mounted on uncoated slides. Normal/hyperplas-tic/dysplastic/cancerous epithelia, were dissected andharvested under a stereomicroscope. Target epithelia wereconfirmed by routine light microscopy of the corresponding4 lm H&E slide avoiding contamination of adjacent non-tar-get tissues. Samples containing <70% target epithelia werenot taken for further analysis. Briefly, DNA was isolatedfrom the harvested epithelia by proteinase K (Merck, Darms-tadt, Germany) treatment and ethanol/salt precipitationmethods. DNA samples were stored at �20 �C until use.

PCR

The DNA preparation (5 lL) was added to the PCR mixture(20 lL) containing 2.5U of Taq DNA polymerase (Promega,Madison, WI, USA), 1 · PCR buffer, 1.5 mmol/L MgCl2,0.2 mmol/L of each deoxynucleotide triphosphate, and1 lmol/L of each primer. The PCR protocol was then carriedout at a PCR system (Eppendorf AG, Hamburg, Germany)with an initial 5-min denaturation step at 94 �C coupled toa repeating cycle of 1 min at 94 �C (denaturation), 30 s at58 �C (annealing), and 30 s at 72 �C (extension) for 36 cy-cles, followed by a final cycle at 72 �C for 10 min. A negativereagent control and a negative sample control were in-cluded in each PCR run. Two different sets of primers,one for the target gene (TAOS1 or EMS1) and the other forthe control gene (GAPDH), were present simultaneously inone reaction vessel. Primers of TAOS1 and GAPDH were de-signed with the PRIMER 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primers for the TAOS1 genewere 50-AAG CAT GTC CGA AAG CAG TC-30 and 50-AAA CTCGGC GAC AGA GTG AG-30, which amplified a sequence of235 bp. Primers for the GAPDH gene were 50-ATC ACT GCCACC CAG AAG AC-30 and 50-TGA CAA AGT GGT CGT TGA

510 J. Xia et al.

GG-30, which amplified a fragment of 385bp. Primers for theEMS1 gene, obtained from the published sequences, were50-TCC CAA TCC AGA GAC CCG-30 and 50-TCC CCT GAT GCCCAG GTC-30, and the amplicon was 113 bp.11

Electrophoresis, sequence analysis andquantitation of PCR products

The resulting PCR products were visualized after electro-phoresis by UV illuminating on a 2.5% agarose gel containingethidium bromide. The EMS1, TAOS1 and GAPDH bands wereidentified by their nucleotide weights comparing to the100 bp DNA ladder (MBI Fermentas, Hanover, USA). In orderto confirm the accuracy of PCR, randomly selected PCRproducts (113 bp and 235 bp) were sent for purificationand sequencing (Dingan Biotechnology Co. Shanghai, China).Each PCR product was sequenced twice in each direction.We obtained the final sequencing reports. Each consensusgene sequence was determined by comparison to gene data-base of Human beings using the BLAST searching tool(http://www.ncbi.nlm.nih.gov/BLAST/) at the NationalCenter for Biotechnology Information (NCBI), USA. The elec-trophoresis images of the PCR products were captured andrecorded by gel imaging analysis system (Quantity 2000,Bio-Rad, USA). A computerized program (Quantity One-4.2.2, Bio-Rad, USA) was used to determine the densitome-try of stained nucleotide bands on the gel. The intensity ofeach band was computerized according to the following for-mula: average optical density · band area. Then the EMS1 orTAOS1:GAPDH ratios of each band were determined. Geneamplification was defined as at least a 2-fold increase inthe EMS1:GAPDH or TAOS1:GAPDH ratios, relative to theaverage ratios obtained with normal tissue DNA. PCR wascarried out at least twice in the positive cases.

Statistical analysis

Chi-square analysis and Fisher’s exact tests were used toestimate statistical difference of gene amplification amongdifferent groups, and to estimate statistical correlations be-tween gene amplification and clinicopathological parame-ters (mean age, gender, tumor site, primary tumor size,histopathological grading, lymph node status and clinicalstage). The analysis of variance (ANOVA) was used to testdifferences of quantitative measurements. Probability va-lue of p < 0.05 (two-sided) was accepted as statistically sig-nificant for all statistical tests carried out using SPSS�

version 10.0 software (SPSS Inc., Chicago, IL, USA).

Table 2 Amplifications of EMS1 and TAOS1 in OLK (n = 30) and p

Groups Total cases (%) EMS1-nona

OLK 30 24(80.0)TAOS1-nonamplified cases 20(66.7) 17(56.7)TAOS1-amplified cases 10(33.3) 7(23.3)

OSCC 33 14(42.4)TAOS1-nonamplified cases 16(48.5) 11(33.3)TAOS1-amplified cases 17(51.5) 3(9.1)

a v2 test, association between TAOS1 and EMS1 amplifications.

Results

TAOS1 and EMS1 amplifications in OLK and primaryOSCC

A total of 78 samples were subjected to identify gene ampli-fication by differential PCR using GAPDH gene as the con-trol. Sequence analysis showed that 113 bp and 235 bpamplicons matched fragment of EMS1 and TAOS1 gene with100% identity, verifying that they were specific amplicons ofEMS1 and TAOS1, respectively. DNA samples obtained fromnormal tissues showed TAOS1:GAPDH ratios of 0.98 ± 0.12(mean ± SD), and EMS1:GAPDH ratios of 0.84 ± 0.16(mean ± SD). Case would be considered amplified if present-ing a TAOS1:GAPDH ratio higher than 1.96, or an EMS1:-GAPDH ratio higher than 1.68. Totally, 25 cases wereEMS1-amplified and 27 cases were TAOS1-amplified. Table2 shows gene amplification results in OLK and primary OSCC.Comparing with EMS1, although TAOS1 amplification fre-quency was higher in leukoplakic epithelia and lower inOSCC tissues, the differences were not statistically signifi-cant (p = 0.815 > 0.05). In OSCC, TAOS1 and EMS1 amplifica-tions showed significant correlation (p = 0.005). In otherwords, cases with TAOS1 amplification tended to displayEMS1 amplification in OSCC patients. Coamplification ofboth genes was demonstrated in 10.0% of OLK cases and42.4% of OSCC cases (Table 2).

Characteristics of TAOS1 and EMS1 amplifications indifferent stages of oral carcinogenesis

During different stages (histopathological grading) of oralcarcinogenesis, gene amplification frequency and trendlineare as indicated in Figure 1. Both TAOS1 (p = 0.001 < 0.05)and EMS1 (p = 0.000 < 0.05) amplifications showed signifi-cant correlations with multistage oral carcinogenesis. Nei-ther normal mucosa nor hyperplastic (non-dysplastic)epithelia showed EMS1 or TAOS1 amplification. TAOS1amplification began from mild-dysplastic epithelia andEMS1 from moderate-dysplastic epithelia. With the diseaseprogressing, TAOS1 amplification frequencies varied from33.3% (3/9), 50% (4/8), 42.90% (3/7) to 35% (7/20), with nostatistical significance among different histopathologicaltypes (p > 0.05). Neither did EMS1 amplification frequenciesshowed significant differences with 0%, 37.5% (3/8), 42.9%(3/7), 40% (8/20) in turn. However, in OSCC with lymph nodemetastasis, both TAOS1 (79.6%, 10/13) and EMS1 (84.6%,11/13) amplification frequencies increased significantly

rimary OSCC (n = 33)

mplified cases (%) EMS1-amplified cases (%) pa

6(20.0) 0.3723(10.0)3(10.0)

19(57.6) 0.0055(15.2)14(42.4)

0

0.2

0.4

0.6

0.8

1

Normal

mucos

a

Hyperp

lasia

Mild

dysp

lasia

Mod

erate

dysp

lasia

Severe

dysp

lasia

OSCC with

out m

etasta

sis

OSCC with

meta

stasis

Stages of oral carcinogenesis

Am

plif

icat

ion

freq

uenc

y

EMS1

TAOS1

p>0.05

p<0.05

A

0

2

4

6

8

10

Normal

mucos

a

Hyperp

lasia

Mild

disp

lasia

Mod

erate

displa

sia

Severe

dysp

lasia

OSCC with

out m

etasta

sis

OSCC with

meta

stasis

Stages of oral carcinogenesis

Coa

mpl

ific

atio

n ca

ses

B

Figure 1 Amplifications of TAOS1 and EMS1 genes in the progress of oral carcinogenesis: (A) amplification frequency of TAOS1 andEMS1 genes in different stages of oral carcinogenesis. Amplification frequency increased significantly in OSCC with lymph nodemetastasis (p < 0.05, v2 test). (B) co-amplified cases in different stages of oral carcinogenesis.

Amplifications of TAOS1 and EMS1 genes in oral carcinogenesis 511

(p < 0.05). Coamplification began from moderate-dysplasticepithelia with the cases ranging from 1, 2, 5 to 9 in courseof oral carcinogenesis, respectively (Fig. 1). Figure 2 displaysrepresentative electrophoresis images of TAOS1 and EMS1genes with different histopathological gradings.

Correlation of TAOS1 or EMS1 amplification withclinicopathological parameters

No significant relationship was found between TAOS1/EMS1amplification and mean age, gender, primary tumor site inOSCC patients (p > 0.05, Table 3). However, both TAOS1and EMS1 amplifications showed significant associationswith T-primary tumor, N-regional lymph node, histologicaltype and clinical stage (p < 0.05, Table 3).

Discussion

Chromosomal band 11q13 is a frequent site of gene amplifi-cation in a variety of human malignancies. Quite recently,

the TAOS1 gene was identified by high-resolution mappingof the 11q13 amplicon in OSCC cell lines. Later, TAOS1was also nominated as ORAOV1(oral cancer overexpressed1).6 TAOS1 and EMS1 are thought as two key genes in driving11q13 amplification.6,12 Oral carcinogenesis is a multistepprocess that results from the accumulation and interplayof multiple molecular genetic events in many chromosomesand genes over the lifetime of cancer.9,13–15 Evidence hashighlighted the importance of CCND1 amplification in oralcarcinogenesis,16–18 however, some opposite reports sug-gested controversy on this point.11 Then, our study providednew insights into the other two target genes, TAOS1 andEMS1, in multistage carcinogenesis of OSCC.

Firstly, of the 33 OSCC cases, 51.5% displayed TAOS1amplification and 57.6% displayed EMS1 amplification in ourstudy. 20% of EMS1 amplification in HNSCC has been re-ported, although only five OSCC cases were included.11 Allthese confirmed that amplifications of both genes could alsobe observed in primary OSCC, in concordance with the resultsof OSCC cell lines. Moreover, coamplification was observed in42.2% of OSCC cases. Five cases were EMS1-amplified and

Figure 2 PCR analysis of TAOS1 (A) and EMS1 (B) geneamplifications from different histopathological-grading DNAsamples in positive cases, if there were. Two different pairsof primers, TAOS1/EMS1 and GAPDH, were present simulta-neously in one reaction vessel. The resulting PCR products werevisualized by UV-illuminating on an ethidium bromide-stained2.5% agarose gel and quantitated by image analysis densitom-etry. M: 100 bp DNA ladder; lane 1 and lane 2: normal mucosa;lane 3: hyperplastic epithelia; lane 4: mild-dysplastic epithelia;lane 5: moderate-dysplastic epithelia; lane 6: severe-dysplasticepithelia; lane 7: OSCC without lymph node metastasis; Lane 8:OSCC with lymph node metastasis.

512 J. Xia et al.

TAOS1-nonamplified and three cases were TAOS1-amplifiedand EMS1-nonamplified, which indicated genetic complexityand diversity in OSCC.9,19 Since there were 33.3% TAOS1-amplified and 20% EMS1-amplified cases in OLK, we deducedthat TAOS1 or EMS1 amplification might be an early eventduring oral carcinogenesis.

In agreement with our hypothesis, TAOS1 and EMS1amplifications paralleled with the progress of oral carcino-genesis, suggesting their potential roles in oral carcinogen-esis. Moreover, TAOS1 might be involved earlier than EMS1.Our results indicated significant correlation between geneamplification and multistage carcinogenesis. In the dynamicmonitoring of oral carcinogenesis, both TAOS1 and EMS1amplifications undergone a consecutive process as follow-ing: nonamplification (normal mucosa and hyperplastic epi-thelia), amplification (mild-dysplastic epithelia, moderate-dysplastic epithelia, severe-dysplastic epithelia and OSCCwithout metastasis), and significant amplification (OSCCwith metastasis). Furthermore, from our data concerningcoamplification, gene amplification seemed to be demon-strating a tendency from no gene amplification (normal mu-cosa and hyperplastic epithelia), single gene amplification(TAOS1 in mild-dysplastic epithelia), to double gene ampli-fication (TAOS1 and EMS1 in moderate-dysplastic epithelia,severe-dysplastic epithelia and OSCC). Further analysis sug-gested that significant association of TAOS1 and EMS1

amplification was not in OLK (p = 0.372) but in OSCC(p = 0.005). That is to say, coamplification tended to morefrequently happen in advanced cancer than in early-stagedisease. All these supported the view that the more ad-vanced a cancer is, the more complex the gene is.19,20

Owing to its genetic diversity, OSCC is a highly heteroge-neous disease. Since clinical symptoms and histopatholo-gical appearance structures result from a series ofbiological events on a cellular and molecular level whichfrequently decide the future malignant potential of a can-cer, it is likely that the result (i.e., metastasis) of theseevents may be assessed more accurately by studying theseevents in the primary cancer itself. Presently, identificationof molecular biomarkers for risk prediction, diagnosis andprognosis in pre-malignant lesions and cancers has becomeone important challenge for cancer research.21–25 Althoughamplification and/or expression status of TAOS1 might be auseful marker for the diagnosis and prognosis of OSCC,6 noclinical research has corroborated this view. Two parame-ters, lymph node metastasis and EMS1 amplification, werestatistically significant independent predictors of a reduceddisease-specific survival and early recurrence in HNSCC.11

Our results showed that TAOS1 and EMS1 amplificationswere associated with increased regional lymph nodemetastasis, poorly differentiated cancers, larger cancerdiameters and advanced clinical stage. Although withoutfollow-up study, we could deduce that patients with TAOS1or EMS1 amplification might have poor prognosis becauseanyone of those significant parameters is commonly thoughtas important indicator for disease recurrence and decreasedsurvival rate in OSCC, that is, poor prognosis.4,21–25

The EMS1 gene encodes the human homologue of cortac-tin, which localizes in the cytoplasm and in areas of the cell-substratum contacts. Tight relationship between EMS1amplification and cortactin levels has been suggested byavailable information.11,12,26 It has become clear that theEMS1 protein is involved in regulating the interactions be-tween components of adherens-type junctions and organiz-ing the cytoskeleton and cell adhesion structures ofepithelia and carcinoma cells. All these indicate that itmay modulate cancer cell invasion and metastasis.7,8,26

However, the cancer-related function of the TAOS1 proteinhas not been well understood, although TAOS1 amplifiedand mRNA overexpressed in 63% OSCC cell lines.6 Therefore,for a long time we focus on determining TAOS1 protein leveland functions. Although TAOS1 antibody could not be com-mercially available until now, we are making progress indi-rectly. Now, by means of RNA interfering technology, thebiological behaviors of some OSCC cell lines are evaluated,such as cell adhesion, cell proliferation, and cell migration.We are trying to make it clear whether cell behaviorchanges resulted from protein changes. From availableinformation till now, we hypothesized that TAOS1 mightparticipate in cell cycle control and regulate cell prolifera-tion, just as CCND1.

In conclusion, TAOS1 and EMS1 appear now to play partroles in oral carcinogenesis and the former might be in-volved earlier than the latter. Both genes might be candi-date biomarkers for diagnosis and prognosis in OSCC.However, prior to their routine use in clinical practice, fur-ther examinations with larger series of patients are neededto implement and confirm the previous results.

Table 3 Association of TAOS1 or EMS1 amplification with clinicopathological characteristics in OSCC

Characteristics Total (n = 33) EMS1-amplified cases (n = 19) pa TAOS1-amplified cases (n = 17) pa

Mean age (yrs) 60.3 57.3 0.104b 57.8 0.246b

Gender 0.698 0.708Male 24 13 13Female 9 6 4

Tumor site 0.881 0.494Tongue 17 10 10Others 16 9 7

T-primary tumorc 0.013 0.007T1 + T2 26 12 10T3 + T4 7 7 7

N-regional lymph node 0.015 0.032Non-metastasis 20 8 7Metastasis 13 11 10

Histological type 0.011 0.013Well 19 7 6Moderately + Poorly 14 12 11

Clinical stagec 0.015 0.032I + II 20 8 7III + IV 13 11 10

a v2 test.b The analysis of variance (ANOVA).c According to TNM staging system of UICC in 1992.

Amplifications of TAOS1 and EMS1 genes in oral carcinogenesis 513

Acknowledgement

This work was supported by grants from the National NaturalScience Foundation of China (No. 30300387, 30471891) andthe 10th 5 Year Plan of National Key Technologies R&D Pro-gram in China (2004BA720A28).

References

1. Lin M, Smith LT, Smiraglia DJ, Kazhiyur-Mannar R, Lang JC,Schuller DE, et al. DNA copy number gains in head and necksquamous cell carcinoma. Oncogene 2006;25:1424–33.

2. Hui AB, Or YY, Takano H, Tsang RK, To KF, Guan XY, et al.Array-based comparative genomic hybridization analysis iden-tified cyclin D1 as a target oncogene at 11q13.3 in nasopha-ryngeal carcinoma. Cancer Res 2005;65:8125–33.

3. Zaharieva BM, Simon R, Diener PA, Ackermann D, Maurer R,Alund G, et al. High-throughput tissue microarray analysis of11q13 gene amplification (CCND1, FGF3, FGF4, EMS1) in urinarybladder cancer. J Pathol 2003;201:603–8.

4. Meredith SD, Levine PA, Burns JA, Gaffey MJ, Boyd JC, WeissLM, et al. Chromosome 11q13 amplification in head and necksquamous cell carcinoma. Association with poor prognosis. ArchOtolaryngol Head Neck Surg 1995;121:790–4.

5. Schuuring E. The involvement of the chromosome 11q13 regionin human malignancies: cyclin D1 and EMS1 are two newcandidate oncogenes–a review. Gene 1995;159:83–96.

6. Huang X, Gollin SM, Raja S, Godfrey TE. High-resolutionmapping of the 11q13 amplicon and identification of a gene,TAOS1, that is amplified and overexpressed in oral cancer cells.Proc Natl Acad Sci USA 2002;99:11369–74.

7. Campbell DH, Sutherland RL, Daly RJ. Signaling pathways andstructural domains required for phosphorylation of EMS1/cortactin. Cancer Res 1999;59:5376–85.

8. Schuuring E, van Damme H, Schuuring-Scholtes E, Verhoeven E,Michalides R, Geelen E, et al. Characterization of the EMS1gene and its product, human Cortactin. Cell Adhes Commun1998;6:185–209.

9. Scully C, Field JK, Tanzawa H. Genetic aberrations in oral orhead and neck squamous cell carcinoma (SCCHN): 1. Carcino-gen metabolism, DNA repair and cell cycle control. Oral Oncol2000;36:256–63.

10. Freier K, Sticht C, Hofele C, Flechtenmacher C, Stange D, PuccioL, et al. Recurrent coamplification of cytoskeleton-associatedgenes EMS1 and SHANK2 with CCND1 in oral squamous cellcarcinoma. Genes Chromosomes Cancer 2006;45:118–25.

11. Rodrigo JP, Garcia LA, Ramos S, Lazo PS, Suarez C. EMS1 geneamplification correlates with poor prognosis in squamous cellcarcinomas of the head and neck. Clin Cancer Res 2000;6:3177–82.

12. Patel AM, Incognito LS, Schechter GL, Wasilenko WJ, SomersKD. Amplification and expression of EMS-1 (cortactin) in headand neck squamous cell carcinoma cell lines. Oncogene1996;12:31–5.

13. Liu SC, Klein-Szanto AJP. Markers of proliferation in normal andleukoplakic oral epithelia. Oral Oncol 2000;36:145–51.

14. Zhang L, Rosin MP. Loss of heterozygosity: a potential tool inmanagement of oral premalignant lesions? J Oral Pathol Med2001;30:513–20.

15. Mehrotra R, Gupta A, Singh M, Ibrahim R. Application ofcytology and molecular biology in diagnosing premalignant ormalignant oral lesions. Mol Cancer 2006;5:11.

16. Fujii M, Ishiguro R, Yamashita T, Tashiro M. Cyclin D1amplification correlates with early recurrence of squamous

514 J. Xia et al.

cell carcinoma of the tongue. Cancer Lett 2001;172:187–92.

17. Mineta H, Miura K, Takebayashi S, Ueda Y, Misawa K, Harada H,et al. Cyclin D1 overexpression correlates with poor prognosisin patients with tongue squamous cell carcinoma. Oral Oncol2000;36:194–8.

18. Miyamoto R, Uzawa N, Nagaoka S, Nakakuki K, Hirata Y,Amagasa T. Potential marker of oral squamous cell carcinomaaggressiveness detected by fluorescence in situ hybridization infine-needle aspiration biopsies. Cancer 2002;95:2152–9.

19. Scully C, Sudbo J, Speight PM. Progress in determining themalignant potential of oral lesions. J Oral Pathol Med2003;32:251–6.

20. SchliephakeH.Prognostic relevanceofmolecularmarkers of oralcancer–a review. Int J Oral Maxillofac Surg 2003;32:233–45.

21. Takes RP, Baatenburg de Jong RJ, Schuuring E, Hermans J, VisSV, Litvinov SV, et al. Markers for assessment of nodalmetastasis in laryngeal carcinoma. Arch Otolaryngol Head NeckSurg 1997;123:412–9.

22. Muller D, Millon R, Velten M, Bronner G, Jung G, Engelmann A,et al. Amplification of 11q13 DNA markers in head and neck

squamous cell carcinomas: correlation with clinical outcome.Eur J Cancer 1997;33:2203–10.

23. Hermsen M, Alonso Guervos M, Meijer G, van Diest P, SuarezNieto C, Marcos CA, et al. Chromosomal changes in relation toclinical outcome in larynx and pharynx squamous cell carci-noma. Cell Oncol 2005;27:191–8.

24. Myo K, Uzawa N, Miyamoto R, Sonoda I, Yuki Y, Amagasa T.Cyclin D1 gene numerical aberration is a predictive marker foroccult cervical lymph node metastasis in TNM Stage I and IIsquamous cell carcinoma of the oral cavity. Cancer 2005;104:2709–16.

25. Rodolico V, Aragona F, Cabibi D, Di Bernardo C, Di Lorenzo R,Gebbia N, et al. Overexpression of cyclin D1 and interactionbetween p27Kip1 and tumour thickness predict lymph nodemetastases occurrence in lower lip squamous cell carcinoma.Oral Oncol 2005;41:268–75.

26. van Damme H, Brok H, Schuuring-Scholtes E, Schuuring E. Theredistribution of cortactin into cell–matrix contact sites inhuman carcinoma cells with 11q13 amplification is associatedwith both overexpression and post-translational modification. JBiol Chem 1997;272:7374–80.