Oral Field Cancerization: Carcinogen-induced Independent ... · Patients with a head and neck squamous cell carcinoma (HNSCC) often develop multiple (pre)malignant lesions. This finding

Review

Oral Field Cancerization: Carcinogen-induced Independent Events orMicrometastatic Deposits?

Monique G. C. T. van Oijen and Pieter J. Slootweg1

Department of Pathology [M. G. C. T. v. O., P. J. S.], and Jordan Laboratory,Department of Haematology [M. G. C. T. v. O.], University Medical CenterUtrecht, 3508 GA Utrecht, the Netherlands

AbstractPatients with a head and neck squamous cell carcinoma(HNSCC) often develop multiple (pre)malignant lesions.This finding led to the field cancerization theory, whichhypothesizes that the entire epithelial surface of theupper aerodigestive tract has an increased risk for thedevelopment of (pre)malignant lesions because of multiplegenetic abnormalities in the whole tissue region.Demonstration of alterations in histologically normaltumor-adjacent mucosa from HNSCC patients supportedthis hypothesis. Currently, the question has been raisedwhether multiple lesions develop independently from eachother or from migrated malignant or progenitor cells.The majority of the mucosal alterations appear to berelated to the exposure to alcohol and/or tobacco.Moreover, almost all primary remote tumors fromHNSCC patients appear to be clonally unrelated.Therefore, there is more evidence that field cancerizationis due to multiple independent events than to migrationof genetically altered cells.

IntroductionEpidemiology of HNSCC. Worldwide, HNSCC2 is the sixthmost common malignancy in men and accounts for approxi-mately 5% of malignant tumors in the population of developedcountries (1–3). However, in parts of Southeast Asia, head andneck cancer is the most common malignancy, accounting for upto 50% of malignant tumors (1, 4). These percentages reflectthe prevalence of the specific risk factors in these geographicregions: tobacco and alcohol in the developed countries andchewing of betel quid in South East Asia (3). Studies of theepidemiology of HNSCC have identified tobacco and alcoholuse as independent risk factors, but when both factors arecombined there is a synergistic effect (3, 5, 6).

Survival of HNSCC patients depends on tumor size, nodal

stage, and success of initial treatment and has not improvedvery much during the last decades (7). In general, a 5-yearsurvival rate of 50% can be obtained, although some anatomicalsites are associated with a less favorable prognosis than others.The prognosis of HNSCC patients is adversely influenced bythe development of second primary tumors (8). The incidencerate of second primary tumors is 10–35%, depending on boththe location of the first primary tumor and the age of the patient(8–10).Different Oral Field Cancerization Theories. The findingthat HNSCC patients often present widespread premalignantlesions and MPTs in their UADT led Slaughteret al. (11) in1953 to postulate the concept of field cancerization. Theyhypothesized that the entire epithelial surface of the UADT hasan increased risk for the development of (pre)malignant lesionsbecause of multiple genetic abnormalities in the whole tissueregion.

The mucosal changes in the entire UADT were generallyconsidered to be the result of exposure to carcinogens thatcaused multiple genetic abnormalities in the whole tissue region(11). The multiple squamous cell lesions described in the oralfield cancerization process were thought to have developedindependently of each other.

An alternative theory for the occurrence of multiple (pre)-malignant lesions has been proposed in the last decade and is basedon the premise that any transforming event is rare and that themultiple lesions arise due to widespread migration of transformedcells through the whole aerodigestive tract (12–14). Two types ofmigration might be involved in the concept of this last theory: (a)migration of tumor cells by, for example, saliva (micrometastases);or (b) intraepithelial migration of the progeny of the initiallytransformed cells (Fig. 1). If metastatic cells from a HNSCCmigrate through the blood or lymph system, they usually settle inthe lung or in the first lymph node encountered. Therefore, thisroute would not lead to tumor deposits in the mucosal surface thatlines the UADT.

Information about all of the different theories can begathered by two different ways of investigation. One way is tosearch for differences in alterations between histologically nor-mal TAM from smokers/alcohol drinkers and normal TAMfrom nonsmokers/nondrinkers. If there are migrating tumorcells, one expects them to be present in TAM from smoking aswell as nonsmoking HNSCC patients. Thus, TAM from smok-ing as well as nonsmoking HNSCC patients should exhibit thesame alterations. These alterations should be absent in smokinghealthy individuals, as in those cases, there is no source formigrating tumor cells. Furthermore, any observed TAM alter-ations should be identical with the alterations in the primarytumor in case of migration of advanced tumor cells, whereas incase of migrating progenitor cells, at least some early tumori-genic alterations would be identical between TAM and theinvasive tumor. In contrast, when there are no migrating cells,TAM changes in smoking HNSCC patients have to be regardedas smoking-induced independent events and therefore should

Received 8/18/99; revised 12/15/99; accepted 12/23/99.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby markedadvertisementinaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.1 To whom requests for reprints should be addressed, at Department of Pathology(H04-312), University Medical Center Utrecht, P. O. Box 85500, 3508 G.A. U-trecht, the Netherlands. Phone: 31-30-2506561; Fax: 31-30-2544990; E-mail:[email protected] The abbreviations used are: HNSCC, head and neck squamous cell carcinoma;MPT, multiple primary tumor; UADT, upper aerodigestive tract; TAM, tumor-adjacent mucosa; EGFR, epidermal growth factor receptor; TGF-a, transforminggrowth factora; LOH, loss of heterozygosity; CDK, cyclin-dependent kinase.

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be absent in TAM from nonsmoking HNSCC patients. More-over, some of the alterations in TAM in smoking HNSCCpatients should also be present in normal mucosa from healthysmokers.

The second way to investigate the different theories is toinvestigate the clonality of the multiple (pre)malignant lesionsby analysis of early genetic alterations in the development ofHNSCC. The separate lesions would share common geneticalterations if they would have developed from a single clone.Clonal relationship between multiple lesions points to migra-tion of tumor cells or progenitor cells. If no clonal relationshipbetween multiple lesions can be found, independent develop-ment of the lesions is more likely. Both pathways above out-lined will be followed in this review article. We will start byreviewing the field changes and their relationship with the mostimportant epidemiological risk factors as far as available datajustify. After evaluating whether these data are in support ofeither the migration or the independent origin theory, we willpay attention to the studies in which clonal relationship betweenMPTs was studied and discuss whether data on clonality ofMPTs support any of both theories: migration of precursor cellsor independent tumor development. Finally, some clinical im-plications of these studies will be mentioned.

Oral Field Changes and Their Relationship with RiskFactorsMorphological Changes. In 1962, Nieburgset al. (15) re-ported malignancy-associated changes within smear cells ofnormal buccal mucosa in patients with malignant disease. Thechanges consisted of an increase in nuclear size, discontinuousnuclear membrane, numerous Feulgen-negative areas, in-creased associated chromatin surrounding the clear areas, and

absence of a single large nucleolus. Inczeet al. (16) confirmedthe increase in nuclear area in normal oral mucosa remote fromHNSCCs using ultrastructural analysis. They also described analtered nuclear to cytoplasmic area ratio.

A reduction in cytoplasmic area was later shown by Ogdenet al. (17, 18). They suggested that tobacco might play a role inthis alteration. However, they could only show a nonsignificanttendency for the influence of tobacco and alcohol on thismorphological change in the HNSCC patients (18). Alternativecausative factors, such as chronic inflammation, irradiation, andchemotherapy, were all excluded, and they speculated aboutan additional influence of the malnutrition that was present inmost patients (17, 18). Cancer-free smokers, though, showed aslight increase in nuclear area compared to cancer-free non-smokers (19).Aneuploidy and Chromosomal Aberrations. In the last dec-ade, other field changes have been reported. Althoughpolyploid cells were not detected in normal tumor-distant mu-cosa (18), aneuploidy was observed in hyperplastic/inflamma-tory mucosa that subsequently developed in an invasive carci-noma (20). This aneuploidy was not detected in hyperplastic/inflammatory mucosa from healthy individuals. Hittelmanet al.(21) determined by using chromosomein situhybridization thatgenomic instability in the upper aerodigestive epithelial fieldincreases the risk to develop a HNSCC.

A whole series of chromosomal aberrations was shown byfluorescencein situ hybridization in cells from brushes frommacroscopically normal cheek opposite the site of the HNSCC(22). Unfortunately, only one patient out of the 10 investigatedpatients did not smoke. This patient did not show chromosomalchanges in the normal cells; however, one patient is obviouslynot enough to draw any conclusion about the role of tobacco inthese chromosomal aberrations. The mucosa from cancer-freecontrol smokers was compared with mucosa from cancer-freenonsmokers. No significant chromosome aneusomies were de-tected, although a trend toward aneusomies of chromosomes 2,6, and Y was observed in the mucosa from smokers (22).

In another study, polysomies of chromosomes 7 and 17were observed in TAM from HNSCC patients (23, 24). Almostall patients smoked, which might suggest that the polysomieswere smoking induced. However, the polysomies were signif-icantly higher in TAM than in tumor-distant mucosa, whichrefutes this idea. A significant loss of chromosome Y wasdetected in TAM from smoking HNSCC patients, but this lossappeared not to be present in the nonsmoking patients (25).

In another publication on chromosomal aberrations inHNSCC patients, the investigators used microsatellite analysis(26). Allelic loss of chromosome 13 was detected in 10 of 16informative TAM samples when they were compared to bloodsamples. No data on a relationship of this finding with smokingcould be detected.Alterations in Cytokeratin Expression. Cytokeratins are theintermediate filament proteins found in the cytoplasm of allepithelial cells. There are at least 20 different keratin polypep-tides that are expressed in different combinations, depending onthe type of epithelium and the degree of differentiation (27).Aberrant expression of cytokeratins has been shown during theprocess of HNSCC carcinogenesis (28, 29). Presence of cytok-eratins 7, 8, 13, 16, and 19 was observed at abnormal anatom-ical sites or at abnormal intraepithelial levels in normal mucosafrom HNSCC patients (30–33).

Only one study is available in which cytokeratin expres-sion was studied in relation to smoking habits. Expression of

Fig. 1. Different field cancerization theories. HNSCC patients frequently de-velop more than one tumor and often have widespread premalignant lesions. Thedifferent theories for the occurrence of these multiple lesions are depicted.

250 Review: Oral Field Cancerization



cytokeratins 7 and 8 in TAM occurred more frequently in thesmoking group of patients than in the nonsmoking group (31).Changes in Blood Group Antigens of the ABH System.Histo-blood group antigens are cell surface carbohydrates thatshow changes in expression related to tissue type, differentia-tion state and cell motility capacity. Type 2 chain ABH-carbo-hydrate structures are distributed broadly in epithelial and en-dothelial cells, independent of the patient’s ABO blood group.In normal oral and laryngeal epithelium, type 2 chain ABH-antigens are expressed on parabasal cells (34). A 4-fold lowerexpression of type 2 chain ABH-antigen was shown in exfoli-ated cells from macroscopically normal mucosa from six dif-ferent places distant from the HNSCC, compared with healthyindividuals (32). Because the ABH type 2 chain expression wasalways lower in the mucosa from the patients than in themucosa from healthy controls, this antigen may be promising asa negative marker for field change and risk indication.Foci of Cyclin D1 Expression. Cyclins are cell cycle regula-tors that are functional only when associated with CDKs. Cy-clin D1 regulates the G1-S transition in the cell cycle and isfunctional when it is associated with either cdk4 or cdk6 (35).Amplification of the chromosome 11q13 region, which resultsin overexpression of the proto-oncogenecyclin D1 has beendescribed in about half of the HNSCC (36, 37).Cyclin D1amplification has been shown in premalignant lesions and theamplification frequency progresses from premalignant lesionsto invasive carcinoma (36). Bartkovaet al. (37) observedclearly defined foci of cyclin D1 expression in sections ofnormal mucosa adjacent to HNSCC that were not seen insections of normal mucosa from healthy individuals. Whetherthis finding relates to smoking habits has not been addressed.Increased Expression of the Epidermal Growth Factor Re-ceptor. One of the cellular oncogenes that play a role in thedevelopment of HNSCC is theEGFR. This gene encodes thereceptor of the growth factors epidermal growth factor andTGF-a. Ligand binding to the extracellular domain of theEGFR causes receptor dimerization, which activates tyrosinekinase function. This leads to autophosphorylation and subse-quent phosphorylation of intracellular target proteins, whichresults in proliferation (38).

EGFR mRNA overexpression, as well as protein overex-pression, has been demonstrated in nearly all HNSCCs (39–41). EGFR expression increases during the development ofnormal epithelium to dysplastic epithelium and further in-creases when dysplasia becomes an invasive carcinoma (42).

Several studies have shown increased expression of theEGFR in TAM (39–46). In five of these studies, an overex-pression of the protein was observed (42–46), one study de-scribed an elevated mRNA level (40), and one study showedamplification of theEGFRgene (45). The EGFR overexpres-sion was almost as high in TAM from nonsmoking/nondrinkingHNSCC patients as in TAM from smoking/drinking HNSCCpatients (43). No increase in EGFR expression was observed inmucosa from cancer-free smokers compared to cancer-freenonsmokers (43). Furthermore, both Grandiset al. (46) andVan Oijen et al. (43) showed that EGFR expression in themucosa from the HNSCC patients was less elevated when theepithelium was located more distant to the tumor (43, 46).These results suggest a paracrine effect on the EGFR expres-sion due to factors released by the tumor and not due toinfluence of smoking.Elevated TGF-a mRNA. Besides investigation of the EGFRalso one of its ligands, TGF-a, was investigated. It was shownthat the mRNA level of TGF-a was 5-fold increased in normal

TAM compared with mRNA levels in control normal mucosa(40), but whether this relates to smoking is unknown.Increased Proliferation. One of the characteristics of a tumoris an increased proliferation. Shinet al. (47) showed a sequen-tial increase in proliferating cell nuclear antigen expression inhead and neck tumorigenesis.

An increased number of proliferating epithelial cells wasalso shown in TAM from HNSCC patients (47, 48). Thisincrease in proliferation was related rather to smoking than tothe presence of a HNSCC, as it was only detected in the TAMfrom smoking HNSCC patients. No increase was observed inTAM from nonsmoking HNSCC patients (49). This increase inproliferating cells was observed not only in TAM from smokingHNSCC patients but also in mucosa of the UADT from healthysmokers (49). A trend toward an increased proliferation wasalso shown in the mucosa from ex-smoking cancer-free andHNSCC patients. Thus, the elevated proliferation persists forsome time after quitting smoking. The higher proliferativeactivity in the epithelium of the UADT from smokers andex-smokers may well constitute a fertile soil for genetic eventsculminating in the development of HNSCC.p53 Overexpression.Loss of function of the tumor suppressorp53 can result in uncontrolled cell division and progressivegenomic instability (50). Abnormalities of thep53 tumor sup-pressor gene are among the most frequent molecular eventsin cancer. More than 90% of the HNSCCs contain mutatedp53 (51), and in 50% of the tumors, LOH ofp53 has beenshown (52).

Mutant p53 has a higher stability than wild-type p53,which allows accumulation to levels detectable by immunohis-tochemistry (53, 54). The frequency of p53-positive cells grad-ually increases as oral epithelium progresses from normal tohyperplasia to dysplasia to carcinoma (55, 56). Focal p53positivity was detected more often in normal TAM than inhealthy control epithelium (56–60). Mutations in thep53genewere identified in both normal TAM and tumor-distant mucosafrom HNSCC patients, in contrast to healthy nonsmokers (58,61, 62). These mutations were polyclonal and differed fromthose detected in the adjacent tumor (58, 61).

Focal overexpression of p53 might reflect an increasedrisk of second primary tumors in these patients. Previously, itwas shown that more p53 positive cell clusters were present inepithelium surrounding multicentric HNSCC as compared tounicentric HNSCC (63). Furthermore, p53 expression abovethe basal cell layers in oral mucosa has been found to be anearly event of malignant transformation and has predictivevalue for the development of HNSCC (60). This notion is incontrast with another study in which the investigators foundthat p53 overexpression in TAM could not predict the likeli-hood of a second primary squamous cell carcinoma (64). How-ever, no distinction was made between basal and suprabasalp53 expression in that study.

The possible role of smoking in inducing p53 overexpres-sion in TAM was examined by immunohistochemistry (57). Nodifferences in uniformly spread overexpression of p53 wereobserved between the mucosa from smoking and nonsmokingHNSCC patients compared to healthy controls.

In contrast to the uniformly spread p53 overexpression,focal overexpression of p53 occurred more frequently in normalepithelium from smoking HNSCC patients than from nonsmok-ing HNSCC patients and from healthy control individuals (57).However, the elevated number of p53 positive cell clusterscould not be detected in mucosa from healthy smokers. Thisindicates that besides the exposure to tobacco, other environ-

251Cancer Epidemiology, Biomarkers & Prevention



mental factors and/or genetic factors must contribute to theabundance of p53 positive cell clusters in TAM. Exposure toalcohol was an additional factor in the HNSCC patients (57)and might have played a role in the development of the p53positive cell clusters together with the influence of tobacco. Inaddition to the findings in tissue sections (57), p53 overexpres-sion and mutations were detected in some clusters of culturedcells from normal mucosa of healthy smokers (62). These dataon p53 gene alterations and p53 protein overexpression inapparently healthy TAM suggest a pivotal role for p53 in theearly phase of oral carcinogenesis.Lack of bcl-2 Expression. In normal tissue, there is a balancebetween cell proliferation and programmed cell death (apopto-sis). Alterations of both pathways contribute to a clonal expan-sion of cancer cells. bcl-2, an apoptosis inhibitor, and its familymembers (among others, bax, an apoptosis inducer) play animportant role in the regulation of the apoptotic pathway (65).Apoptosis itself did not vary significantly in the different stagesof HNSCC tumorigenesis (66). However, there was lack ofbcl-2 expression in HNSCC and in normal TAM compared tocontrol mucosa. No data on relationship with smoking werementioned (66). Because bcl-2 is supposed to inhibit apoptosis,one would expect an increase in bcl-2 expression during tu-morigenesis and therefore the lack of bcl-2 expression is rathersurprising. However, to estimate the bcl-2 activity, the expres-sion of bcl-2 has to be interpreted in the context of levels ofother bcl-2/bax family members.Increased Glutathione S-Transferase.GlutathioneS-trans-ferases are detoxification enzymes that comprise different iso-classes (a-, m-, p-, andt-class), of which the expression levelsare tissue specific and dependent on polymorphisms (67). Glu-tathioneS-transferases are multifunctional, intracellular, solu-ble, or membrane-bound enzymes, which catalyze the conju-gation of many electrophilic hydrophobic compounds with thetripeptide GSH. GlutathioneS-transferasem is an isozyme witha marked specificity for catalyzing the conjugation of epoxides,such as benzo(a)-4,5-oxide and sterene-7–8-oxide, carcino-genic components in cigarette smoke (68). A 2-fold higher riskof laryngeal cancer has been shown among smokers who lackthe glutathioneS-transferasem isoenzyme (69). In contrast,glutathione S-transferases were increased in dysplastic orallesions and HNSCC (70).

The expression of all glutathioneS-transferase isoenzymeswas significantly higher in the suprabasal and superficial layersof normal oral mucosa from HNSCC patients who subsequentlydeveloped a second primary tumor than in normal oral mucosafrom HNSCC patients who were free of disease for at least 7years (71). Also, in cell scrapes of macroscopically normalTAM, elevated levels of glutathioneS-transferasem- andp-class were observed (71). Whether these findings have anyrelationship with smoking habits has not been assessed. Thereason for these increased levels is an intriguing fact, becauseelevated levels of detoxification enzymes protect against car-cinogenic attacks. It may reflect a futile response to the pres-ence of carcinogenic metabolites from tobacco, because allinvestigated individuals smoked. It becomes even more com-plex when one realizes that malignant tissue might benefit fromthe higher levels of glutathioneS-transferase in case of chem-otherapy (70). The reason for the high levels of glutathioneS-transferase may not yet be clear; nevertheless, it seems tohave a predictive value for the development of a (second)primary tumor.Expression of the Proto-oncogene Product eIF4E.Controlof gene expression at the translational level is important in cell

growth and proliferation. A key participant in regulation oftranslation is the proto-oncogene product eIF4E or mRNA 59cap-binding protein (72). This protein has been found to beexpressed at an elevated level in HNSCC (73). Also, histolog-ically normal margins of resected HNSCCs showed overex-pression of eIF4E (73). Relationships with smoking habits werenot investigated.Protein Tyrosine Kinase and Protein Tyrosine PhosphataseActivity. Phosphorylation of proteins on tyrosyl residues is akey mechanism in signal transduction pathways that controlgrowth, differentiation, and cellular architecture of normal andmalignant cells (74). This phosphorylation is strictly regulatedby protein tyrosine kinases and protein tyrosine phosphatases.Normal TAM showed a 2.2-fold increase in protein tyrosinekinase activity compared to the control mucosa from healthyindividuals. In addition, in the TAM, a 1.7-fold elevated ratio ofprotein tyrosine kinase activity to protein tyrosine phosphataseactivity was observed (75). Whether these findings are relatedwith smoking habits is unknown.A Relationship with Tobacco Exposure Has Been Estab-lished for Some TAM Changes.When these data are sum-marized, it appears that TAM contains a lot of alterations,which means that genetic abnormalities are indeed distributedthroughout the epithelium of the entire UADT. Additionalresearch is required to assess which field changes have carci-nogenetic significance and which are merely epiphenomenal.

It seems that TAM from smoking HNSCC patients showsalterations not found in nonsmoking patients (Fig. 2), whichimplies that TAM in smoking patients harbors altered cells thatare absent in nonsmoking patients. If these cells have migratedfrom adjacent tumors, one would expect some changes in TAMfrom nonsmoking patients, which is not supported by thesedata. In addition, some of the field changes observed (increasednuclear area, increased proliferation, and mutations inp53)were already present in healthy smokers without a tumor beingpresent as source of migrating cells (19, 49, 62). Therefore,unless we assume that TAM in both smoking and nonsmokingpatients contains migrating cells that do not betray themselvesby showing as yet unknown alterations, these results supportthe idea that TAM changes in HNSCC patients are carcinogeninduced, independent events rather than being the result ofmigrated transformed cells.

This notion is also supported by the epidemiological find-ing that the risk of developing second primary HNSCCs ishigher in smokers/drinkers than in nonsmokers/nondrinkers (8,9). Moreover, this risk decreases when the patient quits smok-ing and stops abusing alcohol (8).

Clonality StudiesAs outlined in the Introduction, comparing the genetic alter-ations occurring in MPTs of the head and neck area will also behelpful in assessing the strength of either the migration or theindependency theory in explaining oral field cancerization. Ifmultiple tumors develop due to migration of malignant cellsfrom a primary source, then the tumors and dysplasias from thesame patient should show identical genetic alterations, whereasin case of independent origin, these alterations will be different.For these studies, various clonal markers have been used.Clonal Markers. To investigate the relationship betweenMPTs, good clonal markers are needed. To qualify as a marker,such a genetic alteration should (a) occur very early in thedevelopment of the primary lesion, (b) be maintained duringprogression of the lesion, (c) exhibit sufficient variability, and(d) be applicable in the majority of the lesions. We will mention




the genetic markers that have been used to assess the clonalrelationship between separate squamous lesions in the aerodi-gestive tract and discuss whether they meet the above outlined4 requirements.

One of the genetic alterations used is the pattern of X-chromosome inactivation (13). However, when using X-chro-mosome inactivation as marker for investigation of the clonalrelation between tumors lying close to each other, one has torealize that X-chromosome inactivation occurs relatively earlyin embryonic development, resulting in large patches of cellsderived from a common ancestor cell and thus having the sameX-chromosome inactivated (76). In addition, X-chromosomeinactivation can be nonrandom or cell type specific (77).

Karyotypes of tumors have also been used as a clonalmarker (78). However, tissue culture is required for this type ofanalysis, which might cause additional genetic alterations.

Another kind of clonal markers that has been used in-volves LOH patterns for microsatellite markers at differentchromosomal loci (12, 14, 79, 80). LOH at loci on chromosomearms 3p, 9p, and 17p have been shown to occur early incarcinogenesis (12, 80–84). Only a single change might occurin a LOH pattern of about 10 microsatellite markers duringtumor progression and metastasizing (80, 85). However, LOHat loci on chromosomes 3p, 9p, and 17p occurs so frequently(80) that complete loss of all markers may be present in manyof the lesions, which means that the variability of these markersis not high enough to draw conclusions about clonal relation-ships between all lesions.

Finally, p53mutations have been used as a clonal marker(51, 55, 61, 80, 86).p53 mutations are an early event in thedevelopment of HNSCC because they are already present innormal tissue distant from tumors (56, 58, 61), in normal tissuefrom healthy smokers (62), and in premalignant lesions (56).These mutations are diverse enough (80) and stable duringmetastasizing (87). Thusp53 mutations in HNSCC patientsappear to be very useful as clonal marker, whereas the othermarkers are less suitable due to lack of variability and stabilityor due to technical requirements.Polyclonality of MPTs in the Head and Neck. Most studiesthat used clonal markers to investigate the relationship betweenMPTs or to investigate dysplastic lesions occurring in theUADT and that were remote from each other showed poly-clonality between these lesions (14, 79, 80, 86, 88, 89). Only alimited amount of MPTs showed the same genetic alterations as

evidenced by showing identical microsatellite alterations, LOHpatterns, or cytogenetic features (14, 78, 79, 90). However, theoverwhelming majority of remote MPTs show no clonal rela-tionships and can therefore be assumed to have developedindependently.

HNSCC or adjacent premalignant lesions that are locatedvery close to each other more often show identical geneticchanges. Califanoet al. (12) observed a clonal relationshipbetween five HNSCCs and the tumor-adjacent premaligantlesions by using LOH pattern analysis. Two patients withsynchronous HNSCC tumors lying close to each other wereinvestigated for clonality by Van Oijenet al. (80). The firstpatient showed an identicalp53mutation and an identical LOHpattern in both tumors, whereas the other patient did not showidentical aberrations. This strongly suggests that in the firstpatient, migration of malignant cells has occurred. In the nineother patients investigated with HNSCC lying remote fromeach other, no clonality was observed. Thus, in patients inwhom two HNSCCs are not separated by a large distance, sometumors might have developed from the same primary lesion,whereas other tumors have developed independently.

More evidence for the existence of lesions arising inde-pendently in the UADT came from the diversity ofp53 muta-tions identified in histologically normal TAM from HNSCCpatients (58, 61). All identified mutations differed from thep53mutation in the matched HNSCC. This excludes the possibilitythat new lesions in these patients develop by migrated tumorcells, as well as the explanation that early lesions in TAM occurdue to migration of progenitor cells with ap53 mutation.

One precaution needs to be considered. The conclusionthat MPTs develop independently is only tenable when one canexclude the possibility that there is migration of cells with a yetunknown genetic alteration that had arisen prior to all “earlyalterations” investigated. However, it is difficult to imagine thatprogenitor cells, with only a minimal genetic alteration, wouldhave the capacity to migrate intraepithelially over large dis-tances. Neither is it very plausible that such progenitor cellswould be displaced by salivary flow, after which they settleelsewhere for outgrowth, because progenitor cells do not haveany invasive or metastatic potential. Furthermore, it has beenshown that a few “early” transformed cells that are surroundedby normal cells do not succeed in the development of a newlesion (91).

In the bronchial tree, the situation may be different, as

Fig. 2. Smoking-induced field changes.The abuse of tobacco is related to fieldchanges in TAM from HNSCC patients(p53 mutations, chromosome Y loss,increased proliferation, cytokeratin 7and 8 expression, and morphologicalchanges). These smoking-induced fieldchanges might be the beginnings ofnew, independently developing lesions.




Franklin et al. (92) reported a case harboring the samep53mutation in dysplastic bronchial mucosal samples taken fromdifferent locations. Migration ofp53mutated progenitor cells ishighly likely to have occurred in that patient, unless this par-ticular p53 mutation was induced independently in these dif-ferent lesions as a consequence of a specific carcinogen.

Clinical Relevance and ConclusionsThe finding that field changes frequently occur in TAM ofHNSCC patients creates a different view on tumor excisionmargins that contain molecularly altered cells (93, 94). Theconclusion that the margin is tumor positive if it shows thepresence of genetically altered cells does not necessarily holdtrue for every molecular marker used. The molecular markerused has to be specific for that particular tumor. For example,the observation that the samep53 mutation is present in themargin (95) as well as as in the tumor might be useful; however,the detection of overexpression of eIF4E in the margins (73) isnot specific for that tumor and more likely reflects a fieldchange caused by smoking.

Most field changes appear to be induced by smoking,which implies carcinogen-induced field cancerization ratherthan field cancerization due to migrated transformed cells. Wealso conclude that almost no remote multiple tumors developdue to migration of tumor cells. They seem to develop inde-pendently as a result of the continuous carcinogenetic influenceof alcohol and/or tobacco. Therefore, patients with a HNSCCshould be advised to quit smoking to reduce the risk of thedevelopment of MPTs, especially because HNSCC patientsseem to be more susceptible (9, 96) to tumor development thancancer-free smokers.

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2000;9:249-256. Cancer Epidemiol Biomarkers Prev Monique G. C. T. van Oijen and Pieter J. Slootweg Events or Micrometastatic Deposits?Oral Field Cancerization: Carcinogen-induced Independent

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Oral Field Cancerization: Carcinogen-induced Independent ... · Patients with a head and neck squamous cell carcinoma (HNSCC) often develop multiple (pre)malignant lesions. This finding