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Chemoprevention of squamous cell carcinoma of the headand neckJohn M. Wrangle and Fadlo R. KhuriPurpose of review

The aim of this article is to summarize progress in

understanding of the biology of squamous cell carcinoma of

the head and neck and of trials to prevent malignant

conversion of oral premalignant lesions and the

development of second primary tumors in those already

treated for squamous cell carcinoma of the head and neck.

Recent findings

The understanding of squamous cell carcinoma of the head

and neck biology is rapidly evolving. Clinical trials for

chemoprevention are involving more diverse regimens,

following disappointing results of retinoid monotherapy.

In-vitro and animal studies form the rationale for the next

generation of studies, employing combination, synergistic

treatments.

Summary

Based on trial data to date, no recommendation for

intervention with a chemopreventive agent can be made. It

is clear, however, that smoking cessation is an effective

intervention for preventing oral premalignant lesions and

second primary tumors. Promising trials are being

conducted and designed currently. The future of this area of

study will involve rational choice of multidrug regimens

based on current understanding of the biology of squamous

cell carcinoma of the head and neck.

Keywords

chemoprevention, head and neck cancer, oral

premalignant lesions, second primary tumors

Curr Opin Oncol 19:180187. 2007 Lippincott Williams & Wilkins.

Winship Cancer Institute/Emory University, Atlanta, Georgia, USA

Correspondence to Fadlo R. Khuri, MD, Professor of Hematology, Oncology,Medicine, Pharmacology and Otolaryngology, Blomeyer Chair in TranslationalCancer Research, Deputy Director, Clinical and Translational Research, SectionHead, Hematology and Oncology, Winship Cancer Institute, Emory UniversitySchool of Medicine, 1365 C Clifton Road, NE, Atlanta, GA 30322, USATel: +1 404 778 4250; fax: +1 404 778 5520; e-mail: fkhuri@emory.edu

Current Opinion in Oncology 2007, 19:180187opyright Lippincott Williams & Wilkins. Unautho

180Abbreviations13-cRA 1rized 3-cis retinoic acid

COX cyclooxygenase

EGFR epidermal growth factor receptor

GST glutathione S-transferase

HPV human papilloma virus

OPL oral premalignant lesion

OSCC oral squamous cell carcinoma

RAR retinoic acid receptor

SCCHN squamous cell carcinoma of the head and neck

SPT second primary tumor

TGF transforming growth factor 2007 Lippincott Williams & Wilkins1040-8746

IntroductionSquamous cell carcinoma of the head and neck (SCCHN)represents a large, worldwide health burden with approxi-mately 500 000 cases diagnosed annually [1]. This oralpremalignant lesion is the subject of study in manyprimary prevention trials. In the United States, 30 200cases of oral cavity and pharynx cancer and 7800 deathswere estimated for 2000 [2]. Second primary tumors(SPTs) represent a significant risk to these patients, withreported rates of development varying from 1.5 to 7% peryear [3,4]. Because these cancers arise in physicallycompact and anatomically complex sites, morbidity con-tinues to be high despite advances in surgery, radiation,and chemotherapy. With expanding knowledge of theaberrant molecular pathways and stepwise acquisition ofgenetic mutations in dysplastic and malignant clones,rational therapeutic options targeting specific mechan-isms of disease become apparent.

Chemoprevention and field cancerization

Chemoprevention, a concept introduced by Sporn et al.[5], is the use of natural or synthetic chemicals for thereversal, suppression, or prevention of conversion of apremalignant lesion to an invasive form. Chemopreven-tion for head and neck cancer encompasses the synchro-nous and metachronous development of SPTs within thecondemned epithelium of the upper aerodigestive tractand lung. Slaughter et al. [6] introduced the concept offield cancerization in 1953, suggesting that multipleneoplasias may develop within an anatomically and histo-logically related site, whether by migration of a clonallineage to an adjacent site, or by separate clones obtainingcritical genetic insults as a result of similar exposuresto tissues throughout a field. Vikram [7] observed thatSPTs are a major source of morbidity and mortality inreproduction of this article is prohibited.

mailto:fkhuri@emory.edu

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Chemoprevention of squamous cell cancer Wrangle and Khuri 181adequately treated malignancies of the head and neck.Thus, successful therapy of SCCHN must include con-sideration of SPT prevention.

Molecular biology of head and necksquamous cell carcinomaThe understanding of the molecular pathology ofSCCHN is incomplete, though rapidly evolving.

P53

A transcription factor important to cell cycle regulation, theaberrant expression of p53 has been implicated in manycancers. Its locus on chromosome 17p is deleted in up to60% of SCCHNs [8]. Shin et al. [9] identified a stepwiseincrease in the proportion of mucosal biopsies with detect-able p53, from normal to hyperplastic to dysplastic tofrank squamous cell carcinoma. Nees et al. [10] showedthat p53 is overexpressed in normal epithelia distant fromthe site of primary tumors. Santos and colleagues [11]found that the vaccinia-related kinase (VRK1) stabilizesp53 as measured by p53 response proteins.

Cyclooxygenase-2

Cyclooxygenase (COX) 1 is constitutively activated,COX-2 is an inducible enzyme that is overexpressed inmany premalignant and malignant tumors including col-orectal adenomas and adenocarcinomas [12], Barrettsesophagus and esophageal adenocarcinoma [1315],hepatocellular carcinoma [16], gastric carcinoma [17],as well as SCCHN [18,19]. Several groups have investi-gated the mechanistic involvement of COX-2 inSCCHN. Subbaramaiah et al. [20] showed overexpressionof COX-2 in mouse embryo fibroblasts with p53 mutationswhen compared with wild type p53 cells. Kinugasa et al.[21] showed decreased invasiveness of human oral squa-mous cell carcinoma (OSCC) cell lines when treatedwith a selective COX-2 inhibitor, and attributed this effectto downregulation of metalloproteinase 2 (MMP-2)and CD44. Gallo et al. [22] demonstrated a correlationbetween COX-2 expression and tumor microvesseldensity, nodal metastases, and vascular endothelial growthfactor (VEGF) in SCCHN biopsies. Wang et al. [23]demonstrated decreased tumor vascularity and sizefollowing celecoxib administration in nude mice inocu-lated with SCCHN cells.

p16

The tumor suppressor gene p16 encodes an inhibitor ofcyclin-dependent kinase 4 (CDK4). Loss of p16 functioncontributes to uncontrolled cell proliferation. The G1regulator retinoblastoma protein (pRb) is regulated byp16 and cyclin D1, and is lost in many OSCC lines[24,25]. Holley et al. [26] found an association betweencyclin D1 polymorphisms and OSCC in tumor biopsies.Zhang et al. [27] showed that p16 mutations impairp53-imposed G1 arrest. Estimates of the frequencyopyright Lippincott Williams & Wilkins. Unauthwith which p16 abnormalities occur vary by study anddegree of histologic abnormality [2830]. Zhang et al. [31]poignantly demonstrated a discrepancy between 68SCCHN biopsies and nine SCCHN cell lines in frequencyof alterations in p16: 10% and 44% respectively.

Transforming growth factor a and epidermal growth

factor receptor

Epidermal growth factor receptor (EGFR) and one of itsligands, transforming growth factor (TGF)a, are known toenhance cellular proliferation in a variety of cancers.Grandis and Tweardy [32,33] demonstrated increasedlevels of EGFR and TGFa mRNA in SCCHN tumorbiopsies and the surrounding normal mucosa when com-pared with normal mucosa. Several groups have reportedeffects of various interventions on TGFa and EGFRexpression. Beenken et al. [34] showed that the vitaminA analogue 13-cis retinoic acid (13-cRA) decreased TGFaexpression in tumor biopsies analyzed before and afterintervention. Endo et al. [35] transfected an antisenseTGFageneintosubcutaneousSCCHNxenografts innudemice and observed decreased TGFa expression andincreased apoptosis within tumors. He et al. [36] achievedsimilar effects using antisense EGFR genes. Using theselective retinoic acid receptor (RAR) agonist LGD1069,Song et al. [37] demonstrated decreased TGFa, EGFR andproliferation in SCCHN cell lines. Masuda et al. [38] usedthe green tea constituent, epigallocatechin-3-gallate todecrease VEGF activity in SCCHN cell lines, attributingthis to inhibition of nuclear factor-kb and STAT3 activity.

Retinoid biology

In 1925 Wolbach and Howe [39] observed much higherrates of lung cancer and upper aerodigestive tract malig-nancies in cattle deprived of dietary vitamin A. Since thattime, much effort has been invested in understanding thepathophysiological underpinning of this observation.Administration of isotretinoin upregulates RAR-b expres-sion [40] and decreases TGFa expression [34]. Whilemany studies have demonstrated aberrant expression ofRAR-a and b in SCCHN, the carcinogenic mechanismappears to be distinct from that of acute promyelocyticleukemia, wherein there is a fusion of promyelocytic zincfinger (PLZF) and RAR-a in t(15,17) [41].

Human papilloma virus

Several cancers have proven etiologic relationships toviruses, notably cervical cancer and human papillomavirus (HPV). Based on the analysis of 36 tumors, Sleboset al. [42] found that 1535% of SCCHNs contain HPVDNA. Using microarray and PCR they found p16, p18,CDC7 genes and several transcription factors to be sig-nificantly overexpressed in HPV-positive compared withHPV-negative tumors. HPV may be an etiologic agent ina subset of SCCHN and may have important prognosticimplications [43].orized reproduction of this article is prohibited.

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182 Head and neckOther genes

Many genes and gene products have been shown to beoverexpressed in SCCHN including bcl-1 [44], S100A7(psoriasin) [45], eIF4e [46,47], the p53 homologue AIS[48], opioid growth factor receptor (OGFr) [49], andglutathione S-transferase (GST)-p [50]. Weed et al.[51] correlated MUC4 retention with better outcomes.Loss of E-cadherin and p27 expression is an early step inSCCHN tumorigenesis [52]. The epigenetic hyper-methylation of SOCS-1 was associated with SCCHN,presumably via complex mechanisms affecting STAT3activation [53].

Estimation of risk and surrogate endpointbiomarkersDeveloping models to accurately predict who willdevelop SCCHN is critical for the treatment of at riskindividuals and possibly for selection of candidates forchemoprevention trials. Lee et al. [54] created a tool formodeling individual risk of SCCHN employing medicalhistory, histology, and molecular markers. Tobacco useand alcohol are the most potent risk factors [4], but ahistory of these exposures is inadequate criteria forselecting high risk individuals. The vast majority ofindividuals who combine these agents will not developSCCHN, and genetic variations have been implicated inan individuals risk [5558].

Many factors predispose tissue toward malignancy, butLippman and Hong [59] assert that individual molecularmarkers are inadequate to determine who will progress toSCCHN with sufficient accuracy. One mechanism bywhich identical exposures to tobacco and alcohol mayresult in different outcomes is polymorphism in themechanisms of metabolizing toxic substances. Tobaccoproduct carcinogens undergo detoxification via cyto-chrome p450 and conjugation, especially by GST,GSTM1 and GSTT1. Whereas previous studies foundGSTM1 and GSTT1 to be associated with developmentopyright Lippincott Williams & Wilkins. Unautho

Table 1 Summary of randomized, control chemoprevention trials in

Study Study agent (dose)

Hong, 1986 [72] 13-cRA (12 mg/kg/day for 3 months)

Stitch, 1988 [76] Vitamin A (200 000 IU/week for 6 months)

Lippman, 1993 [77] 13-cRA (1.5 mg/kg/day for 3 months) then13-cRA (0.5 mg/kg/day for nine months)or b-carotene (30 mg/day for nine months)

Sankaranarayanan,1997 [79]

Vitamin A (300 000 IU/week for 12 months)or b-carotene (360 mg/week for 12 months)

Liede, 1998 [80] Vitamin E (50 mg/day indefinitely), b-carotene(20 mg/day indefinitely), or both

Chiesa, 2005 [83] Fenretinide (200 mg/day for 52 weeks)

13-cRA, 13-cis-retinoic acid.of SCCHN [6062], the CYP system was not. Individualswho possess the null phenotype of these genes, thusunderexpressing them, are at significantly higher riskfor developing SCCHN and SPTs [63,64,65]. Onerecent metaanalysis confirmed the association of GSTM1and GSTT1, and suggests further association withGSTP1, and even CYP1A1 [66].

Loss of heterozygosity (LOH) at three locations onchromosome 3p and at 9p21 was shown to associate withSCCHN by microsatellite polymorphism deletion map-ping [67,68]. Mao et al. [69] found that reversal of LOH at3p14, 9p21, and 17p13 may be markers of response tochemoprevention efforts. Other promising molecularmarkers include cyclin D1, HIF-a, and MUC4[26,51,70,71].

Clinical trialsClinical trials of chemoprevention in SCCHN can bedivided into two categories: primary prevention of malig-nant transformation of oral premalignant lesions (OPLs)(Table 1), and prevention of SPT in those who haveundergone presumably curative treatment of SCCHN(Table 2). Leukoplakia (Fig. 1) and erythroplakia areconsidered OPLs. Statistics regarding rates of spon-taneous regression and malignant conversion are scarce,but in general, erythroplakia has a much higher likelihoodof progressing to SCCHN and leukoplakia of spon-taneous regression. Endpoints for OPL trials are ratesof spontaneous regression and progression to malignancy.In SPT trials, endpoints include rates of development ofSPT and overall survival.

Oral premalignant lesion chemopreventiontrialsOPL chemoprevention trials, while showing earlypromise, have been hampered by drug toxicity and somedisappointing results.rized reproduction of this article is prohibited.

oral premalignant lesions

Patients randomized Outcome

44 13-cRA reverses dysplasia and decreasestumor size

54 Vitamin A reverses dysplasia and preventsnew leukoplakias from forming

70 Low-dose 13-cRA is superior to b-carotenein stabilization or regression of lesions

Long-term follow up showed no differencein cancer free survival [78]

160 Either regimen is superior to placebo forinducing regression of leukoplakia

409 No regimen was superior to placebo atpreventing the development of leukoplakia

137 Early indication of prevention of malignanttransformations but discontinued due tolow recruitment

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Chemoprevention of squamous cell cancer Wrangle and Khuri 183

Figure 1 Leukoplakia

This oral premalignant lesion is the subject of study in many primaryprevention trials. Reproduced with permission from http://www.dent.ohio-state.edu/oralpath2/nospecivic.htm.Vitamin A, retinoids, b-carotene, and vitamin E

At similar times, two groups reported positive effects ofretinoids, or vitamin A and its analogues, on leukoplakia.In a phase III trial, Hong et al. [72] randomized 44 patientswith leukoplakia to receive 13-cRA (12 mg/kg for3 months) or placebo. In the treatment arm, 67% oflesions decreased in size and 54% showed reversal ofdysplasia versus 10% in both categories among thosereceiving placebo. Stich et al. [7376] reported significantrates of leukoplakia remission and suppression offormation of new lesions among fisherman in Kerala,India, when administered vitamin A (100 000 IU/day)and b-carotene (180 mg/day) when compared withplacebo. Because of dose related toxicity inherent tocertain retinoids, alternate retinoid-receptor, subtype-specific retinoids and dosing strategies have beenpursued. Lippman et al. [77] treated patients withleukoplakia with high dose isotretinoin (1.5 mg/kg/dayopyright Lippincott Williams & Wilkins. Unauth

Table 2 Summary of randomized, control chemoprevention trials o

Study Study agent (Dose) P

Hong, 1990 [87] 13-cRA (50100 mg/m2 for12 months)

Bolla, 1994 [89] Etretinate (50 mg/day for one monththen 25 mg/day indefinitely)

Jyothirmayi, 1996 [90] Retinyl palmitate (200 000 IU/day)

EUROSCAN, 2000 [91] Vitamin A (300 000 IU/day for one year,then 150 000 IU/day for one year), orN-acetylcysteine (600 mg/day), or both

ROG and UTMDACC,2006 [92]

13-cRA (30 mg/day for 3 years)

13-cRA, 13-cis-retinoic acid; ROG, Radiation Oncology Group; SPT, seconCenter.for 3 months) then randomized them to receive low-doseisotretinoin (0.5 mg/kg/day for 9 months) or b-carotene(30 mg/day for nine months) and found the low doseisotretinoin arm to be superior in stabilizing or causingregression of lesions. Unfortunately, in long-term followup, no significant differences in cancer-free survival wereseen [78]. Also in Kerala, India, Sankaranarayanan et al.[79] found both retinyl acetate (300 000 IU/week for12 months) and b-carotene (360 mg/week for 12 months)to be superior to placebo in inducing spontaneousregression of leukoplakia. In contrast, Liede et al. [80]saw no statistically significant differences betweenpatients receiving vitamin E, also known as a-tocopherol,(50 mg/day), b-carotene, a dimer of retinol (20 mg/day),both, or placebo. Two groups have concluded thatfenretinide (200 mg/day), a synthetic retinoid, hasactivity in leukoplakia and is well tolerated [81,82].While a phase III study of fentretinide in resectedOPL indicated some activity in preventing malignantprogression, the study was discontinued due to lowrecruitment [83].

Other agents in oral premalignant lesions

Armstrong et al. [84] conducted a phase I study ofBowmanBirk inhibitor, a soybean derived serine pro-tease inhibitor. In a randomized study of ketorolac, anonselective COX inhibitor, versus placebo in leuko-plakia, Mulshine et al. [85] found no difference incomplete or partial response. Rudin et al. [86] showedtolerability of a mouthwash containing ONYX-015,an attenuated adenovirus cytotoxic to cells with p53mutations, with reversal of dysplasia seen in multiplepatients in this small trial.

Second primary tumor chemoprevention trialsEarly OPL trials suggested a role for chemopreventionof SPT.

First-generation trials

Following promising initial results of retinoids in OPLs,Hong et al. [87] initiated a trial to see if similar therapyorized reproduction of this article is prohibited.

f second primary tumors

atients randomized Outcome

103 No effect on the primary tumor, butsignificantly fewer SPTs

316 No advantage over placebo in preventionof SPTs

93 No advantage over placebo in preventionof SPTs

2592 No differences in overall survival, event-freesurvival, or rate of SPT formation

1190 Low-dose 13-cRA is ineffective at reducingthe rate of SPT or death. Smoking cessationis effective for preventing SPTs [93]

d primary tumor; UTMDACC, University of Texas, MD Anderson Cancer

http://www.dent.ohio-state.edu/oralpath2/nospecivic.htmhttp://www.dent.ohio-state.edu/oralpath2/nospecivic.htm

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184 Head and neckcould prevent SPT after surgery/radiotherapy forSCCHN. One hundred and three patients were random-ized to receive 13-cRA (50100 mg/m2 orally) or placebofor 12 months. During the course of the trial there wereno differences in the frequency of local, nodal, ordistant recurrence, but there were significantly fewerSPTs (4% versus 24% in the placebo arm after 32 monthsof follow up, P 0.005). Several patients experienceddose-limiting toxicity. After 54.5 months, the differencebetween the treatment and placebo group remainedstatistically significant, although diminished fromprevious levels [88].

Bolla et al. [89] randomly assigned 316 treated cases ofSCCHN (stage I and II: surgery with or without radio-therapy) to receive etretinate (50 mg/day for 1 monthfollowed by 25 mg/day orally) or placebo for 24 months.After 41 months median follow up, no statisticallysignificant difference was found between the twogroups.

Jyothirmayi et al. [90] randomized 106 patients whoachieved complete regression of their SCCHN to retinylpalmitate (200 000 IU/day) or placebo and found nostatistically significance difference in rates of SPT orlocal recurrence.

Second-generation trials

The European Organization for Research and Treatmentof Cancer (EUROSCAN) conducted a factorially designedstudy [91] of vitamin A (300 000 IU/day for 1 yearfollowed by 150 000 IU/day for 1 year), N-acetylcysteine(600 mg/day), both, or placebo. After median followup of 49 months for 2592 randomly assigned patients,no statistically significant difference was observed inoverall survival, event-free survival, or developmentof SPT.

The Radiotherapy Oncology Group and the Universityof Texas MD Anderson Cancer Center conducted apivotal study [92] of stage I and II SCCHN involving1190 patients, randomly assigned to receive either lowdose 13-cRA (30 mg/day) or placebo for 3 years. Whilelow-dose isotretinoin was ineffective at reducing the rateof SPT or death, analysis of the data echoed previousreports: smoking cessation is the most effective methodof preventing SPT in SCCHN [93].

Two additional studies with smaller patient populationshave recently reported no improvement in the rate ofSPT among patients treated with 13-cRA (0.5 mg/day) orantioxidant vitamins [94]. Bairati et al. [95] reported asignificantly increased rate of SPT during administrationof vitamin E versus placebo, and a decreased rate of SPT,though not statistically significant, after discontinuationof therapy.opyright Lippincott Williams & Wilkins. UnauthoShin et al. [96] tested the tolerability of a multidrugregimen in a phase II trial of treated SCCHN patients.Patients received 13-cRA (50 mg/m2/day orally), a-toco-pherol (1200 IU/day orally), and IFN-a (3 106 IU/m2subcutaneous injection 3/week). The proportion of5-year disease free survival in this patient populationwas 79% and the regimen was well tolerated. Phase IIItesting of this regimen is currently underway.

DiscussionThe most important consideration in prevention ofSCCHN has been and will continue to be modificationof risk factors. Tobacco users must know the dangers theyface, and be assisted in their efforts to quit. Even for thosealready diagnosed with SCCHN, smoking cessation willresult in improved outcomes. The future of SCCHN andSPT chemoprevention lies in the development of welltolerated, multiagent regimens. Multiple agents havebeen studied and shown to have activity in SCCHN.Farnesyl transferase inhibitors decrease the enzymaticactivation of H-ras, which is mutated in 527% ofSCCHNs, and are currently under phase I/II investi-gation in SCCHN and lung cancer [9799]. As discussed,COX-2 is overexpressed in SCCHN, and has beenimplicated in several mechanisms of carcinogenesisincluding apoptosis and angiogenesis. EGFR inhibitorsare currently under study in SCCHN trials. Theobservation by Zhang et al. that EGFR and COX-2,inhibited simultaneously, yield synergistic activityagainst SCCHN xenografts in nude mice may haveimplications for chemoprevention [100]. ONYX-015,the adenovirus cytotoxic to p53 mutant-containingcells has completed phase I/II testing, and its role inchemoprevention is being evaluated [86]. Shin et al. arestudying a three agent combination of 13-cRA, IFN-a,and a-tocopherol.

ConclusionThe future of SCCHN chemoprevention lies in combi-nations of agents, the selection of which will be based uponour evolving understanding of the molecular basis ofSCCHN. Biomarkers, and risk modeling will be essentialfor assessment of prognosis and response to treatment.

References and recommended readingPapers of particular interest, published within the annual period of review, havebeen highlighted as: of special interest of outstanding interestAdditional references related to this topic can also be found in the CurrentWorld Literature section in this issue (p. 267).

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Chemoprevention of squamous cell carcinoma of the head andneckIntroductionChemoprevention and field cancerization

Molecular biology of head and neck squamous cell carcinomaP53Cyclooxygenase-2p16Transforming growth factor and epidermal growth factor receptorRetinoid biologyHuman papilloma virusOther genes

Estimation of risk and surrogate endpoint biomarkersClinical trialsOral premalignant lesion chemoprevention trialsVitamin A, retinoids, -carotene, and vitamin EOther agents in oral premalignant lesions

Second primary tumor chemoprevention trialsFirst-generation trialsSecond-generation trials

DiscussionConclusionReferences and recommended reading