Advances and applications of oral cancer basic research

Review

Advances and applications of oral cancer basic researchq

Sabrina Daniela da Silva a,b, Alfio Ferlito c, Robert P. Takes d,!, Ruud H. Brakenhoff e,MeV Dominguez Valentin f, Julia A. Woolgar g, Carol R. Bradford h, Juan P. Rodrigo i,j, Alessandra Rinaldo c,Michael P. Hier a, Luiz P. Kowalski baDepartment of Otolaryngology-Head and Neck Surgery, Jewish General Hospital, McGill University, Montreal, CanadabDepartment of Head and Neck Surgery and Otorhinolaryngology, Centro de Tratamento e Pesquisa Hospital do Cancer A.C. Camargo, São Paulo, BrazilcENT Clinic, University of Udine, Udine, ItalydDepartment of Otolaryngology-Head and Neck Surgery, Radboud University Nijmegen Medical Center, Nijmegen, The NetherlandseDepartment of Otolaryngology-Head and Neck Surgery, VU University Medical Center, Amsterdam, The NetherlandsfDepartment of Oncology, Clinical Sciences, Lund University, Lund, SwedengOral Pathology, School of Dental Sciences and Dental Hospital, University of Liverpool, Liverpool, UKhDepartment of Otolaryngology-Head and Neck Surgery, University of Michigan, Ann Arbor, MI, USAiDepartment of Otolaryngology, Hospital Universitario Central de Asturias, Oviedo, Spainj Instituto Universitario de Oncología del Principado de Asturias, Oviedo, Spain

a r t i c l e i n f o

Article history:Received 19 February 2011Received in revised form 1 July 2011Accepted 5 July 2011Available online xxxx

Keywords:Oral cancerSquamous cell carcinomaMolecular biologyCancer researchAdvances and applications

s u m m a r y

Cancer of the oral cavity accounts for almost 3% of cancer cases in the world. The incidence varies widelyreflecting geographic differences in exposure to risk factors. The recent rise in younger age groups andfemales seen in many countries is of particular concern. Treatment and management of complications,locoregional recurrence and further primary tumors result in high morbidity and mortality especiallywhen the disease is advanced stage at initial diagnosis. Progress in cancer research has provided abun-dant new knowledge about cellular processes and molecular biology underlying oral carcinogenesisand tumor progression. The present review attempts to summarize the current most widely-usedresearch approaches and their application in the prevention, diagnosis, effective treatment, and improvedoutcome of oral cancer.

! 2011 Elsevier Ltd. All rights reserved.

Introduction

Although the incidence of oral cavity cancer is not well docu-mented since it is unfortunately often grouped with oropharyngealsubsites, it is thought to be the 8th most frequent cancer in theworld among males and the 14th among females,1 accounting fornearly 3% of all cancer cases.2 Squamous cell carcinoma originatingin the mucosal linings accounts for more than 90% of oral cavitycancers.3,4 The highest incidence rates occur in Pakistan, Brazil, In-dia, and France.1 The main risk factor for oral cancer is exposure toexogenous carcinogens such as tobacco smoke and alcohol. Annu-ally, it is estimated that 127,459 deaths are caused from oral cavitycancer worldwide, of which 96,720 occur in developing countries.2

Oral squamous cell carcinoma (OSCC) is characterized by invasiveand frequent perineural growth, a considerable rate of early recur-

rences, and frequent lymph nodes metastasis. Often these patientsdevelop second primary cancers in the same or adjacent anatomicalregion. Many patients are advanced-stage at diagnosis and incur sig-nificantmorbidity andmortality due to the disease itself and the sub-sequent clinical management with its complications.5–8 Advances incancer researchhaveprovided abundantnewknowledge about cellu-lar processes andmolecular biology inOSCC. Our knowledge of carci-nogenesis, identification of biological markers, and molecularly-targeted therapies is advancing through basic research, translationalresearch and clinical trials, and ultimately analysis of factors specificto the individual andtheir tumormayresult ineffective ‘‘personalizedmedicine’’.9 In the following sections, recent advances and applica-tions of OSCC research and their impact on prevention, diagnosis,effective treatment, and improved prognosis are considered.

Research in risk factors for oral cancer

OSCC is a multi-causal disease with close interrelationshipsamong etiologic factors. Risk factors include lifestyle habits (tobac-co exposure and alcohol consumption), dietary factors, occupa-tional activity, socioeconomic status, exposure to external agents,

1368-8375/$ - see front matter ! 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.oraloncology.2011.07.004

q This paper was written by members and invitees of the International Head andNeck Scientific Group (http://www.IHNSG.com).! Corresponding author. Address: Department of Otolaryngology-Head and Neck

Surgery, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HBNijmegen, The Netherlands. Tel.: +31 24 3613508; fax: +31 24 3540251.

E-mail address: [email protected] (R.P. Takes).

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Please cite this article in press as: da Silva SD et al. Advances and applications of oral cancer basic research. Oral Oncol (2011), doi:10.1016/j.oraloncology.2011.07.004

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and genetic susceptibility.10–14 It is probable that there are addi-tional causative factors yet to be identified.

The deleterious effects of tobacco use and excessive alcoholconsumption are well-known.10,15 There are approximately 1.1 bil-lion smokers worldwide with 80% living in developing countries.The prevalence of OSCC in cigarette smokers is 4–7 times greaterthan in non-smokers.16,17 In Southeast Asia, areca nuts (Arecacatechu), inverted smoking and smokeless tobacco (snuff) are addi-tional, important risks.18–20 Although tobacco and alcohol are inde-pendent risk factors, they have a synergistic effect in combinationwith a clear dose dependent correlation between duration and fre-quency of exposure and tumor development.21,22 Ethanol increasespermeability of the mucosa permitting the action of nitrosamines,hydrocarbons, and acetaldehyde. Smoking increases the acetalde-hyde burden following alcohol consumption, and the alcohol con-sumption enhances the activation of pro-carcinogens by inductionof cytochrome P450-2E1-dependent microsomal biotransforma-tion system in the mucosa.23 Carcinogenic agents may directlycause mutations in the DNA but also suppress the DNA repair en-zymes (the critical component against human cancer). Brennanet al.24 presented evidence linking cigarette smoking to specificp53 mutations but the exact mechanism of carcinogenesis is notalways clear.10,15,21,22

Human papillomavirus (HPV) could also be considered to berelated to life style and it is strongly associated with the develop-ment of oropharyngeal cancers. The role of HPV in the develop-ment of OSCC is less well established and probably involves onlya small minority of cases, generally estimated around 5%.11,25,26

Unfortunately, some authors consider cancers arising from the oralcavity and oropharynx together as ‘‘oral cancer’’. This is reflected indifferences in reported incidence figures. Whereas HPV is very rel-evant and frequent in oropharynx cancer, often treated with(chemo)radiotherapy, it is uncommon and less relevant in oral can-cers which are treated surgically.27,28

Genetic susceptibility (predisposition) to OSCC is significantespecially in young patients29–33 and based on inherited differ-ences in the efficiencies of metabolizing carcinogens, DNA repair,and cell cycle control, alone or in combination. Identification ofindividuals with specific polymorphisms predisposing for cancer,would impact directly on primary and secondary prevention, earlydetection, follow-up strategies and genetic counseling. Singlenucleotide polymorphisms (SNPs) lead to phenotypic effects byseveral mechanisms, including enhanced or reduced transcription,altered post-transcriptional activity or changes in protein struc-ture.34 In addition, polymorphisms related to metabolic enzymespotentially involved in activation (phase I) and detoxification(phase II) of chemical carcinogens may modify susceptibility toOSCC35 by altered protein expression and function.36 Candidatepolymorphisms have been evaluated in DNA repair, cell cycle,xenobiotic metabolism, and growth factor pathways.

The SNP A/G870 in the CCND1 gene (that encodes Cyclin D1) hasbeen associated with OSCC susceptibility. The AA genotype may in-crease 2.38 times the risk for oral cancer.37 However, in anotherstudy, it was the GG wild-type genotype, not the AA genotype, thatwas associated with increased susceptibility (3.37 times).38

Genetic polymorphism in the conjugating enzyme UDP-glucu-ronosyltransferase 1A1 (UGT1A1) and UGT A7 are associated withthe risk of head and neck cancer. UGT1A1 is an enzyme that catal-yses the glucuronidation of the endogenous substrate bilirubin, butalso of the tobacco smoke carcinogens like benzopyrene. Thisraises the question whether the glucuronidation of these carcino-gens is most relevant or a protective function of bilirubin. Bilirubinhas been considered to be only a toxic waste product of hemoglo-bin degradation, but recent findings have shown that bilirubin is apotent antioxidant, which may play a protective role against can-cer. Polymorphism of UGT1A1, which results in lower serum levels

of the endogenous antioxidant bilirubin, was associated with an in-creased risk of head and neck cancer.39 UGT1A7 is an enzyme in-volved in the metabolism of carcinogens present in tobaccosmoke. Genetic polymorphisms in UGT1A7, with predicted alteredenzyme activity, may have a risk-modifying effect on head andneck carcinogenesis.40

Familial aggregation of oral cancer, possibly with an autosomaldominant mode of inheritance, was reported in a very smallpercentage of patients but the responsible genes are unknown.41

A germline p16 mutation segregated with cancer predispositionin a single family with increased head and neck cancer risk. Thusit is likely that the mutant p16 (p16R87P) is implicated in headand neck squamous cell carcinoma (HNSCC) tumorigenesis.42 Amost prominent predisposing genetic factor is a mutation in oneof the Fanconi anemia genes, which act in a complex DNA repairsystem involved in homologous recombination.43 Patients withFanconi anemia are characterized by congenital malformations,bone marrow failure and cancer predisposition, most prominentlyacute myeloid leukemia and SCCs, particularly in the oral cavity.The increased risk for Fanconi anemia patients to develop HNSCCis 500–1000!44 and the accumulated life time risk is estimatedas >30%.45 In patients that are screened regularly it is expected thattumors are diagnosed and treated at an earlier stage. However ran-domized control screening trials are lacking and are in fact uneth-ical. Downstaging in screened vs non-screened groups might givean indication on the benefit of early diagnosis, but these data arenot yet available.

Genome-wide association studies have been successful in iden-tifying common genetic variation involved in susceptibility to eti-ologically complex disease. Recently such a study identifyingcommon genetic variation involved in susceptibility to upperaero-digestive tract (UADT) cancers has been published.46

The identification of a (genetic) risk profile for individuals to de-velop HNSCC and OSCC in particular may not only lead to betterunderstanding of OSCC but also to improved counseling and clini-cal decision making on treatment and follow-up.

Research in oral carcinogenesis and progression models

Oral carcinogenesis, like in other cancers, is a multistep processrequiring the accumulation of multiple genetic alterations thatmodify the normal functions of proto-oncogenes and tumorsuppressor genes which affect cell-cycle regulation, cellular differ-entiation, proliferation and death, DNA repair, and cellular immu-nity.47–57 The molecular mechanisms that underlie the onset ofOSCC and lead to a cellular phenotype showing increased cell pro-liferation, loss of cell adherence, local tissue infiltration, and regio-nal and distant metastasis are increasingly elucidated.47,58 Theseprocesses have been progressively clarified and encompass at leastthe p53 and pRB pathways as well as the transforming growth fac-tor b (TGFB) and PI3K/AKT pathways.59 Moreover, recent studieshave found key signaling pathways and molecules involved withmetastasis mechanisms that include receptor tyrosine kinases,TGFB superfamily, WNT, NOTCH, hedgehog pathway, and NFjB.60

The goal of many studies in molecular detection and diagnosticstrategy is early identification of pre-malignant and malignant le-sions as well as the use of patterns of biomarkers (‘‘biological fin-gerprints’’) in order to prognosticate and risk-stratify patients, andpredict treatment response to conventional therapeutics.61

Genetic changes in OSCC

Chromosomal aberrations such as deletions, amplifications, andstructural rearrangements are hallmarks of malignancy and areseen in head and neck tumors (Table 1).

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There is a relatively common pattern of DNA allelic loss duringthe progression from premalignant to malignant phenotype49 suchas acquisition of specific chromosomal losses at chromosome arms3p, 9p, 17p, and mutations in TP53 (Tables 1 and 2). In comparativegenomic hybridization studies, one of the most promising areasunder investigation is a copy number gain on chromosome 3qand a loss of chromosome 3p which are found at high frequencysuggesting these regions may harbor oncogenes and tumor sup-pressor genes important for the initiation or progression of headand neck cancer62–71(Table 1). An early and common genetic eventin oral premalignancy with potential value in early diagnosis andtumor surveillance is loss of heterozygosity (LOH) in 9p21 in dys-plasia (30%) and OSCC (70–80%).61,72 The detection of 3p and 9ploss by LOH analysis using comparative normal DNA (commonlyperipheral blood lymphocytes) is currently being used in aNational Cancer Institute (http://www.cancer.gov/clinicaltrials)sponsored trial of erlotinib in the prevention of OSCC (EPOC trial).9

Microsatellite or SNP panels to assess LOH are not yet commer-cially available but in the future, LOH testing may become routineand improve OSCC survival by early diagnosis and prediction of tu-mor recurrence.49

Carcinogen exposure can cause simultaneous genetic defectsthroughout the UADT epithelium, putting the epithelium at highrisk for development of premalignant lesions at different stagesof carcinogenesis.73 The concept of ‘‘field cancerization’’, a charac-teristic of head and neck cancers, was introduced in 195374 basedon the hypothesis that prolonged exposure to carcinogens leads tothe independent transformation of epithelial cells at multiple sitesin the adjacent mucosa. The aggregation of genomic alterationsduring progression is assumed to occur in a wide population ofcells, a heterogeneous ‘‘field of genetically altered cells’’ that ismight give rise to a visible precursor lesion. This theory attemptsto explain the high frequency of local recurrences and the emer-gence of second primary tumors in patients with OSCC. This theoryhas been confirmed in many retrospective studies using geneticmarkers. Current data show that approximately 30% of the oraland oropharyngeal cancer cases are surrounded by large fields ofcells with cancer-associated genetic changes that indicate a clonalrelation to the invasive carcinoma.75 These fields frequently re-main behind when the tumor is excised causing secondary tumorsthat are clinically assigned as local recurrences and second primarytumors depending on the distance and time related to the index tu-mor.48,49,76–79 What the clinical relevance of these observations is,needs to be determined but identification of such fields eventuallymay have implications for adjuvant treatment and intensity of fol-low-up.

Metastasis is a complex process requiring tumor cells to pro-gress through multiple stages, governed by successive changes inexpression of certain genes or alterations of gene structures and en-coded products. It begins with cell disassociation within the pri-mary tumor and in OSCC, generally results in metastasis withinregional (cervical) lymph nodes.7,80,81 Identification of biologicalparameters associated with regional metastasis may provide addi-tional information on themetastatic behavior of tumors andmay behelpful in clinical decision making on the treatment of the neck.82

Given the complexicity of the metastatic process, high-through-put techniques are promising tools for predicting regional metasta-sis in oral cancer.83 Discovering differences in gene expressionmight identify critical genes for the process of metastasis. In thisfashion, gene signatures associated with nodal metastasis havebeen identified.82,83 Tumor heterogeneity does not seem to be alimiting factor in the predictive value of these signatures. Themetastatic potential of tumors seems to be encoded in the bulkof a primary tumor, thus challenging this previously mentionedtheory that metastases may arise from rare cells within a primarytumor that have the ability to metastasize.84

Table 1Common chromosome regions aberration in head and neck carcinomas.

Chromosome Chromosome region – alteration

1 Loss 1p36.32 Loss 2q35, 2q363 Loss 3p13–14, 3p21, 3p25; gain 3q25-ter4 Loss 4q25, 4q31–325 Loss 5q21–22; gain 5p6 Loss 6q13, 6q257 Loss 7q31; gain 7p118 Loss 8p21, 8p22, 8p23; gain 8q22, 8q23-ter9 Loss 9p21

10 Loss 10q23, 10q2611 Loss 11q22.2–22.3; gain 11q1312 Gain 12p12.2–1313 Loss 13q14.314 Gain 14q31–32.215 Gain 15q1516 Gain 16q23–2417 Loss 17p13; gain 17q24–2418 Loss 18q; gain 18p19 Gain 19q20 Loss 20p11.2; gain 20q21 Loss 21q11.1, 21q21, 21q22.222 Loss 22q13

Table 2Common gene alterations and potential biomarkers in oral carcinomas.

Markers Function Significance/association

TP53 (p53) Cell-cycle regulation Decreased overall survivalCDKN2A (p16) Senescence, cell-cycle progression Decreased overall survivalCDKN1A (p21) Cell-cycle regulation TumorigenesisCDKN1B (p27) Cell-cycle progression Poor prognosisMDM2 Cell-cycle regulation TumorigenesisMGMT Promoter methylation Decreased overall survivalEGFR Cell proliferation, growth Nodal metastases; more rapid clinical course, consideration for targeted therapyERBB2 Cell proliferation, growth More rapid clinical courseRARB Cell growth and differentiation Decreased overall survivalMYC Cell growth, apoptosis Tumor progressionBCR-ABL1 Cell-cycle regulation and differentiation Tumor progressionRAS Signaling, growth Poor prognosisCCND1 Cell-cycle regulation Nodal metastases; more rapid clinical courseSTAT-3 Cytokine signaling, cell proliferation Decreased survivalVEGF Angiogenesis Consideration for targeted therapyEBV Cell-cycle regulation Diagnostic/screeningHPV Cell-cycle regulation, apoptosis Improved prognosis/local control

Source: http://www.ncbi.nlm.nih.gov.

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http://www.cancer.gov/clinicaltrials

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For the much debated issue of treatment of the clinically N0neck, implementation of these signatures in the clinic could impactdecision making.85 This will be relevant in T1 and T2 oral cancer inparticular since in larger tumors the neck will usually have to beentered for vascular anastomosis of free flap reconstructions.Moreover, depth of invasion may also influence the prior chanceon nodal metastasis and could also be taken in consideration. Any-way, these signatures should first be validated in multicenter set-tings and other procedures like sentinel node mapping will need tobe considered in a decision model for implementation. Sentinellymph node procedures in particular are becoming increasinglyrelevant and due to recent developments like the use of RT-PCRfor detection of tumor cells, the procedure becomes more reliableand more convenient.86,87 The combination of the use of imaging,sentinel node procedures and biological information may providecomplementary information to obtain the most accurate informa-tion on the nodal status of patients.

Development of distant metastasis after initial treatment ofOSCC is not considered a common event but it is associated withfatal outcome.7 Markers for distant metastasis may act as prognos-tic indicators and may play a role in patient counseling and clinicaldecision making as well.

Oncogenes and tumor suppressor genes in OSCC

Studies have explored the common feature of genomic instabil-ity in cancer and identified oncogenes and tumor suppressor geneswhich are correlated in different cancer types.88 The identificationand characterization of genes involved in carcinogenesis will helpto advance drug development and clinical management. Manyexamples can be found in other tumors models, but OSCC studieshave shown up-regulation in key molecules and biochemical path-ways affecting tumor development and progression (Table 2). Theevidence of the role of many of these candidate cancer genes hasrecently been summarized.59

Aberrant activation of the ErbB family members have beenimplicated in many human cancers89–92, including head and neckcarcinomas93–98 and it has been significantly correlated with in-creased tumor progression and metastasis.89,92–94,99 During oralcarcinogenesis, growth signaling is dysregulated through increasedlevels of growth factor receptors and/or their ligands, which pro-mote autocrine stimulation.89,100 This family comprises 4 members(ErbB1/EGFR/HER1, ErbB2/neu/HER2, ErbB3/HER3, and ErbB4/HER4) that play crucial roles in cell proliferation, differentiation,adhesion and migration as well as tumor invasion and apopto-sis.90–92,98 Epidermal growth factor receptor (EGFR) is a widelystudied oncogene in head and neck tumors. As an oncogene itshould be activated either by mutation or amplification. Mutationsare rarely found. Amplification is seen in 30% of the cases.101 Dataon overexpression only are often unreliable. It may be a promisingmarker and prognosticator.73

Bentzen et al.102 demonstrated by dose-fractionation regime(continuous hyperfractionated accelerated radiotherapy – CHART)that EGFR overexpression is a predictive marker for a good thera-peutic response. EGFR continues to be an important area in re-search. Monoclonal antibodies against this receptor and oraltyrosine kinase inhibitors have been studied in multiple phases IIand III clinical trials to determine their efficacy in the non-surgicaltreatment of head and neck cancers and survival benefit of additionof EGFR targeting to radiotherapy has been proven.9,103 However,since oral cancer is primarily treated surgically, these findings willnot have implications in the treatment of oral cancer.

The pathways commonly activated in OSCC are mitogen acti-vated protein kinase, WNT, and PI3K/AKT/mTOR.104–115 Overex-pression of MYC and RAS gene families has an important role intumor progression of HNSCC and has been correlated with poor

prognosis (Table 2)116 In the same way, abundant expression ofcyclins, especially cyclin D1 (CCND1) is a common (36–66%) featureof OSCC and pre-malignant lesions.117–119 CCND1 is a key regulatorcapable of initiating the G1–S transition in the cell cycle regulatedby cyclin-dependent kinases (CDKs).22 CCND1 gene amplificationmay predict worse prognosis and a greater risk of occult cervicallymph nodes metastasis in head and neck tumors.117,118

Another important area involves tumor suppressor genes thatprevent cells from acquiring malignant characteristics and usuallyact in regulating discrete checkpoints during cell cycle progression,monitoring DNA replication and mitosis.22 Inactivation of tumorsuppressor genes can occur via epigenetic or genetic mechanisms.The reasons underlying this choice of gene inactivation routes dur-ing tumorigenesis have not been clarified. Chemical carcinogens intobacco smoke may contribute to the genetic mutations in TP53.120

The inactivation of the TP53 tumor suppressor signaling pathway isseen in most human cancers including OSCC (Table 2).121 The aber-rant p53 protein activity may be caused by mutations in the TP53sequence producing truncated or inactive mutant proteins, or byaberrant production of other proteins that regulate p53 activity(such as gene amplification of MDM2 or viral proteins). Recentstudies have also suggested that inherited genetic polymorphismsin the p53 pathway influence tumor formation, progression, and/orresponse to therapy.121 In the same way, the expression of p16INK4A

protein encoded by the CDKN2A suppressor gene is negative or lowin up to 83% of OSCCs and up to 60% of pre-malignant lesions(Table 2). Several studies have shown frequent CDKN2A gene muta-tions or the frequent loss of gene expression in oral lesions sug-gesting that it is an early step in oral carcinogenesis.13

High-throughput genotyping is being utilized in many tumortypes to probe known oncogene and tumor suppressor gene muta-tions across large numbers of human tumor samples. This ap-proach has the ability to accelerate oncogene and tumorsuppressor gene identification. Results, once obtained, offer greatpotential for identification and targeting of key pathways impli-cated in tumor progression to guide rational strategies for thera-peutic intervention. Work of this nature is underway in head andneck cancer that may identify novel ‘‘drug’’ targets.122

The use of some biomarkers and gene detection panels (Tables 1and 2), some more likely than others, may become a reality in rou-tine clinical management of OSCC. Correlations with certainhistopathological and clinical features have been described,although often inconsistently. Although additional research is nec-essary to lend further insight into the molecular basis of OSCC,these markers and gene expression profiles may become importantas prognostic or predictive indicators and tools for assessment ofevents like regional metastasis, recurrences or early diagnosisand should eventually lead to better outcomes.

Cancer stem cell theory

Unlike the random or ‘‘stochastic’’ model in cancer research,which holds that nearly any cancer cell has the potential to forma tumor, the cancer stem cell model is one of a hierarchical organi-zation, with pluripotent cancer stem cells able to recreate all of thecomponents of the original tumor. Recent reports have shown thatHNSCC contain a subpopulation of cells, called cancer stem cellsthat can self-renew and produce differentiated cells that formthe bulk of the tumor.123 These cells represent a small tumorigenicsubpopulation with distinct phenotype to be more resistant to che-motherapy and radiotherapy than normal cancer cells. The stemcell properties can be identified by a surface marker, such asCD44 and CD24.124 Researchers demonstrated that HNSCC tumorcells that expressed CD44 were able to grow new tumors, whilethe cells that did not express CD44 were not. The tumors that grewwere identical to the original tumors and contained cells that





expressed CD44, as well as cells that did not. This ability to bothself-renew and produce different types of cells is a hallmark ofstem cells. Recent studies have shown aldehyde dehydrogenase 1to be a marker of cancer stem cells in HNSCC.125,126 However, todate the markers available to identify cancer stem cells remainrather specific for the tissue of origin than for the cancer stem cellper se. A generic marker has not been found so far.127 Currenttreatment for HNSCC may selectively kill the more differentiatedcancer cells and produce tumor regression while sparing the cancerstem cells, which can lead to tumor regrowth and relapse. Under-standing what controls the maintenance of stem cells and differen-tiation signals may give insights into the cellular signals involvedin cancer, and may ultimately lead to new approaches to differen-tiation therapy. Thus studies are needed to address how to specif-ically target tumor stem cells in order to eradicate tumors andprevent their recurrence. Despite evidence pointing to the exis-tence of cancer stem cell-like cells in a growing number of cancers,the theory is still a concept and remains controversial.127

Research in clinical prevention and diagnosis

Optical techniques

Toluidine blue and Lugol’s iodine have been used as clinical aidsto identify occult mucosal abnormalities and to demarcate the ex-tent of a potentially malignant lesion prior to excision.128–133

When applied topically or as an oral rinse, toluidine blue bindsto DNA and can help identify malignant lesions with reasonableaccuracy.134 Furthermore, false-positive stains are too frequentfor use as a valid screening tool in primary care settings.133 In addi-tion, controversy exists regarding the subjective interpretation ofmucosal staining and criteria for positive results.135,136 In conclu-sion, no convincing evidence is available to support the use ofthese adjunctive techniques.

Acetic acid-induced whitening of oral mucosa has been pro-posed to enhance and highlight dysplastic lesions137, similar toits use on cervical mucosa.138 ViziLite™ (ViziLite system – ZilaPharmaceuticals, Phoenix, AZ) is one commercially available toolthat makes use of 1% acetic acid-induced whitening of oral tissuesfollowed by examination under diffuse chemiluminescent blue/white light (wavelength of 490–510 nm).138,139 Acetic acid re-moves the glycoprotein barrier and slightly desiccates the mucosa,the abnormal cells then absorb and reflect the blue/white light dif-ferently to normal cells.135,136,140,141 Most investigations have eval-uated highly subjective parameters such as brightness, sharpness,and texture, and, not surprisingly, the findings are inconsistentand contradictory with poor discrimination between keratotic,inflammatory, malignant or potentially malignant white lesions.

Recently, visual autofluorescence (autofluorescence spectros-copy) has been tested in the mouth with promising results that itcan distinguish normal tissues from tumors. The system consistsof a small optical fiber that produces various excitation wave-lengths and a spectrograph that receives and records on a com-puter and analyzes, via dedicated software, the spectra ofreflected fluorescence from the tissue.132 This technique has theadvantage of eliminating subjective interpretation and can providediagnosis in real-time, non-invasively, and in situ.141 The method isyet to be refined, and currently cannot determine tumor depth orhistological grade. Moreover, it is poor at detecting early lesionsand demarcating large lesions as the optical fiber can sample onlya small mucosal area.135,136,140

Narrow band imaging (NBI) is an endoscopic technique usingnarrow-band spectrum optical filters to enhance the visualizationof mucosal and submucosal microvascular patterns. The techniqueis based on the fact that the depth of penetration of light is depen-

dent on its wavelength. The filters used in NBI select blue andgreen light with wavelengths of 415 and 540 nm, respectively, cor-responding to the peaks of absorption of hemoglobin. These fil-tered wavelengths penetrate the superficial layers of mucosa,thus highlighting the capillary network, and at deeper levels en-hance submucosal vessels. In this way, superficial mucosal lesionsthat would be missed by standard white light (WL) endoscopy, arebetter identified in view of their neoangiogenic pattern. Addition-ally, the best image definition for both conventional WL and NBIendoscopy is achieved using a high definition television (HDTV)camera, which provides 1080 lines of resolution, thus allowing asignal definition that is 4.26 times better than standard definitiontelevision. The application of this new technology in the diagnosticwork-up of patients with OSCC (and also with oropharyngeal SCC)was evaluated in a recent study.142 The sensitivity of this techniquein detecting OSCC was 96%, with a specificity of 100% and an over-all accuracy of 97%. The authors confirm the utility of NBI in pre-and intraoperative settings, with better definition of superficialextension of the lesion, detection of synchronous tumors, and iden-tification of unknown primaries. Moreover, the authors found thatHDTV NBI also played a relevant role during follow-up with earlydetection of persistences, recurrences, and metachronous tumors.The specificity obtained by NBI-HDTV underscores the potentialof this technology in the diagnosis and follow-up of OSCC.

Another recent development in the detection of tumor depositsis the use of near infrared fluorescence imaging.143 Fluorescentmolecules labeled to tumor specific proteins or antibodies or othertumor specific probes could make real-time visualization of tumortissue during surgery possible. The use of this technique could helpto obtain adequate surgical margins and could lead to better localcontrol and oncological outcomes without sacrifice of functionallyimportant normal surrounding tissue. The technique can also beused for sentinel node procedures as an alternative for or comple-mentary to radionuclides. The main challenges are to develop spe-cific probes with sufficient tumor to normal tissue ratios and thedevelopment of optimal optical devices to visualize the generatedsignals.

Molecular techniques

The use of saliva and plasma in detection of tumors includingdistant metastases is being investigated.144,145 These sources havebeen used to identify epigenetic changes of hypermethylation spe-cifically of promoters of tumor suppressor genes p16, MGMT,RARb, E-cadherin, and DAPK. Genomic and proteomic studies of tu-mor tissues, plasma, and saliva have identified several promisingcancer signatures (Tables 1 and 2) of potential diagnostic value.In addition, methylation array analysis using saliva has identifiedhighly methylated gene loci with diagnostic and predictive value,with a reported sensitivity of 62–77% and a specificity of 83–100% for OSCC.145 Studies on saliva and exfoliated cells to predictOSCC or recurrence include detection of p53 mutations, LOH of3p and 9p, and HPV with differences in mRNA, microRNA, DNAmethylation, and microsatellite instability profiles.145 Saliva has acluster of protein, secretome, which permits the use of tumormarkers that circulate in blood. In this way, high levels of ErbB2(c-erbB-2/HER2) and cancer antigen 15-3 (CA15-3) were found insaliva in women with breast cancer compared with a low quantityin healthy women.146 Using subtractive proteomics, Hu et al.145 re-vealed several salivary proteins at differential levels between OSCCand matched control subjects. Expectations for future use of saliva-like substrate diagnosis are still evolving.

Another important issue is deciding which potentially malig-nant, intra-epithelial lesions will progress to invasive cancer. Theavailable dysplasia grading systems are incapable of reliablypredicting malignant progression and additional molecular





information could enhance clinical decisions on treatment and fol-low-up.147 Ploidy analysis or DNA copy numbers may provide suchinformation.148–150 The presence or absence of these aberrations inresection margins may be predictive of recurrences.150 Moreover, acombination of the new optical techniques and molecular informa-tion may provide more accurate information.151

Research in treatment and prognostication

The basic prognostic factors in OSCC are encompassed in theTNM classification system: tumor size (T), regional nodal involve-ment (N) and the presence or absence of distant metastasis (M).Although the system is imperfect152, partly because tumors withsimilar morphology and stage may behave differently due to theirdiffering biological characteristics, it is widely used in treatmentplanning, prognostication, and comparison of outcomes.153 How-ever, in the future, it seems likely that biomarkers (Table 2) willsupplement or even replace traditional prognosticators. A varietyof molecular tumor markers have been studied in the clinic fortheir potential to predict disease outcome or response to therapyin OSCC. However, none of these markers appears to provide defin-itive prognostic or predictive information. Additionally, it is unli-kely that any one molecular factor determines the completebehavior of a tumor, and that the complex interaction among onco-genes and tumor suppressor genes cannot be ascertained throughthe analysis of a few molecular markers. A more comprehensivescreen of the molecular defects in head and neck squamous cellcarcinoma obtained through microarray analysis has revealed thatthe molecular classification of these tumors was a better predictorof disease-free survival than clinical and pathological parame-ters154, and that specific gene expression signatures were associ-ated with prognosis in OSCC.155 These findings suggest thatmicroarray technology could provide a novel system of classifica-tion of OSCC. It could be used as an auxiliary tool in the classifica-tion of specific clinical categories of disease and the improvementof specific treatment modalities and patient outcome.

Most OSCCs exhibit limited responsiveness to chemotherapyinvolving cytotoxic drugs, due to mechanisms that either blockintracellular transport of these agents or interfere with their intra-cellular molecular targets.156 In OSCC in particular, surgery re-mains the primary treatment modality of choice, except forinoperable cases. A better understanding of the molecular and bio-logical profiles of OSCC, and the molecular heterogeneity of the dis-ease, could facilitate the development of more efficient targetedtherapies. Most traditional anticancer drugs directly interfere withmitosis, DNA synthesis, and repair systems. A new class of agentsinduces tumor growth retardation (cytostasis) and apoptosis byexploiting aberrant tumor stroma (as membrane-bound receptorkinases), protein dynamics, tumor vasculature, microenvironment,and cellular signaling mechanisms. Drugs that target these path-ways have already entered clinical practice.9157 Nevertheless, sinceOSCC is predominantly a locoregional problem, at least in its earlystages, surgery will likely remain as an important initial treatmentwith therapies like molecular targeting and gene therapy reservedfor the adjuvant or palliative setting.

Conclusions

Oral cavity cancer is an important cause of morbidity and mor-tality, especially in developing countries, and its prevalence maycontinue to rise for the foreseeable future.

Advances in our understanding of OSCC biology and clinical andlaboratory technologies, offer unprecedented prospects for transla-tional research. Implementation in clinical practice of the results ofthis research may enable us to improve early diagnosis, staging and

prognostication, and better select appropriate treatment and fol-low-up regimens. Risk profiles for malignant progression of intra-epithelial lesions could be used to tailor surveillance. Other profilesmay be useful for clinical staging and treatment selection andcould be used for more individualized treatment and this mayeventually lead to overall better oncological and functional out-comes. Nearest to clinical application at this moment are probablythe optical techniques to enhance visualization of neoplastic cellsin vivo. However, at this stage most identified putative relevantbiological markers need to be validated before actual implementa-tion in daily clinical practice may be considered.

Conflict of interest statement

We wish to declare that the submitted work is original and hasnot been submitted or published elsewhere. Also, all authors haveread and approved the manuscript and agree with the current sub-mission. Finally, there are no potential conflicts of interest.

Acknowledgments

This work was supported by Fundação de Amparo à Pesquisa doEstado de São Paulo (FAPESP 06/61039-8 and CEPID/FAPESP98/14335). Silva SD was supported by a FAPESP fellowship(06/61040-6).

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