Oral Field Cancerization: Carcinogen-Induced Independent Events Or Micrometastatic Deposits?

Vol. 9, 249–256, March 2000 Cancer Epidemiology, Biomarkers & Prevention 249

Review Oral Field Cancerization: Carcinogen-induced Independent Events or Micrometastatic Deposits?

Monique G. C. T. van Oijen and Pieter J. Slootweg1 stage, and success of initial treatment and has not improved Department of Pathology [M. G. C. T. v. O., P. J. S.], and Jordan Laboratory, very much during the last decades (7). In general, a 5-year Department of Haematology [M. G. C. T. v. O.], University Medical Center survival rate of 50% can be obtained, although some anatomical Utrecht, 3508 GA Utrecht, the Netherlands sites are associated with a less favorable prognosis than others. The prognosis of HNSCC patients is adversely influenced by the development of second primary tumors (8). The incidence Abstract rate of second primary tumors is 10–35%, depending on both Patients with a head and neck squamous cell carcinoma the location of the first primary tumor and the age of the patient (HNSCC) often develop multiple (pre)malignant lesions. (8–10). This finding led to the field cancerization theory, which Different Oral Field Cancerization Theories. The finding hypothesizes that the entire epithelial surface of the that HNSCC patients often present widespread premalignant upper aerodigestive tract has an increased risk for the lesions and MPTs in their UADT led Slaughter et al. (11) in development of (pre)malignant lesions because of multiple 1953 to postulate the concept of field cancerization. They genetic abnormalities in the whole tissue region. hypothesized that the entire epithelial surface of the UADT has Demonstration of alterations in histologically normal an increased risk for the development of (pre)malignant lesions tumor-adjacent mucosa from HNSCC patients supported because of multiple genetic abnormalities in the whole tissue this hypothesis. Currently, the question has been raised region. whether multiple lesions develop independently from each The mucosal changes in the entire UADT were generally other or from migrated malignant or progenitor cells. considered to be the result of exposure to carcinogens that The majority of the mucosal alterations appear to be caused multiple genetic abnormalities in the whole tissue region related to the exposure to alcohol and/or tobacco. (11). The multiple squamous cell lesions described in the oral Moreover, almost all primary remote tumors from field cancerization process were thought to have developed HNSCC patients appear to be clonally unrelated. independently of each other. Therefore, there is more evidence that field cancerization An alternative theory for the occurrence of multiple (pre)- is due to multiple independent events than to migration malignant lesions has been proposed in the last decade and is based of genetically altered cells. on the premise that any transforming event is rare and that the multiple lesions arise due to widespread migration of transformed Introduction cells through the whole aerodigestive tract (12–14). Two types of Epidemiology of HNSCC. Worldwide, HNSCC2 is the sixth migration might be involved in the concept of this last theory: (a) most common malignancy in men and accounts for approxi- migration of tumor cells by, for example, saliva (micrometastases); mately 5% of malignant tumors in the population of developed or (b) intraepithelial migration of the progeny of the initially countries (1–3). However, in parts of Southeast Asia, head and transformed cells (Fig. 1). If metastatic cells from a HNSCC neck cancer is the most common malignancy, accounting for up migrate through the blood or lymph system, they usually settle in to 50% of malignant tumors (1, 4). These percentages reflect the lung or in the first lymph node encountered. Therefore, this the prevalence of the specific risk factors in these geographic route would not lead to tumor deposits in the mucosal surface that regions: tobacco and alcohol in the developed countries and lines the UADT. chewing of betel quid in South East Asia (3). Studies of the Information about all of the different theories can be epidemiology of HNSCC have identified tobacco and alcohol gathered by two different ways of investigation. One way is to use as independent risk factors, but when both factors are search for differences in alterations between histologically nor- combined there is a synergistic effect (3, 5, 6). mal TAM from smokers/alcohol drinkers and normal TAM Survival of HNSCC patients depends on tumor size, nodal from nonsmokers/nondrinkers. If there are migrating tumor cells, one expects them to be present in TAM from smoking as well as nonsmoking HNSCC patients. Thus, TAM from smoking as well as nonsmoking HNSCC patients should exhibit the Received 8/18/99; revised 12/15/99; accepted 12/23/99. same alterations. These alterations should be absent in smoking The costs of publication of this article were defrayed in part by the payment of healthy individuals, as in those cases, there is no source for page charges. This article must therefore be hereby marked advertisement in migrating tumor cells. Furthermore, any observed TAM alter- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ations should be identical with the alterations in the primary 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- tumor in case of migration of advanced tumor cells, whereas in trecht, the Netherlands. Phone: 31-30-2506561; Fax: 31-30-2544990; E-mail: case of migrating progenitor cells, at least some early tumori- [email protected]. genic alterations would be identical between TAM and the 2 The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; invasive tumor. In contrast, when there are no migrating cells, MPT, multiple primary tumor; UADT, upper aerodigestive tract; TAM, tumor- adjacent mucosa; EGFR, epidermal growth factor receptor; TGF-␣, transforming TAM changes in smoking HNSCC patients have to be regarded growth factor ␣; LOH, loss of heterozygosity; CDK, cyclin-dependent kinase. as smoking-induced independent events and therefore should

absence of a single large nucleolus. Incze et al. (16) confirmed the increase in nuclear area in normal oral mucosa remote from HNSCCs using ultrastructural analysis. They also described an altered nuclear to cytoplasmic area ratio. A reduction in cytoplasmic area was later shown by Ogden et al. (17, 18). They suggested that tobacco might play a role in this alteration. However, they could only show a nonsignificant tendency for the influence of tobacco and alcohol on this morphological change in the HNSCC patients (18). Alternative causative factors, such as chronic inflammation, irradiation, and chemotherapy, were all excluded, and they speculated about an additional influence of the malnutrition that was present in most patients (17, 18). Cancer-free smokers, though, showed a slight increase in nuclear area compared to cancer-free nonsmokers (19). Aneuploidy and Chromosomal Aberrations. In the last decade, other field changes have been reported. Although polyploid cells were not detected in normal tumor-distant mucosa (18), aneuploidy was observed in hyperplastic/inflammatory mucosa that subsequently developed in an invasive carcinoma (20). This aneuploidy was not detected in hyperplastic/ inflammatory mucosa from healthy individuals. Hittelman et al. (21) determined by using chromosome in situ hybridization that genomic instability in the upper aerodigestive epithelial field increases the risk to develop a HNSCC. A whole series of chromosomal aberrations was shown by fluorescence in situ hybridization in cells from brushes from Fig. 1. Different field cancerization theories. HNSCC patients frequently de- macroscopically normal cheek opposite the site of the HNSCC velop more than one tumor and often have widespread premalignant lesions. The different theories for the occurrence of these multiple lesions are depicted. (22). Unfortunately, only one patient out of the 10 investigated patients did not smoke. This patient did not show chromosomal changes in the normal cells; however, one patient is obviously be absent in TAM from nonsmoking HNSCC patients. More- not enough to draw any conclusion about the role of tobacco in over, some of the alterations in TAM in smoking HNSCC these chromosomal aberrations. The mucosa from cancer-free patients should also be present in normal mucosa from healthy control smokers was compared with mucosa from cancer-free smokers. nonsmokers. No significant chromosome aneusomies were de- The second way to investigate the different theories is to tected, although a trend toward aneusomies of chromosomes 2, investigate the clonality of the multiple (pre)malignant lesions 6, and Y was observed in the mucosa from smokers (22). by analysis of early genetic alterations in the development of In another study, polysomies of chromosomes 7 and 17 HNSCC. The separate lesions would share common genetic were observed in TAM from HNSCC patients (23, 24). Almost alterations if they would have developed from a single clone. all patients smoked, which might suggest that the polysomies Clonal relationship between multiple lesions points to migra- were smoking induced. However, the polysomies were signif- tion of tumor cells or progenitor cells. If no clonal relationship icantly higher in TAM than in tumor-distant mucosa, which between multiple lesions can be found, independent develop- refutes this idea. A significant loss of chromosome Y was ment of the lesions is more likely. Both pathways above out- detected in TAM from smoking HNSCC patients, but this loss lined will be followed in this review article. We will start by appeared not to be present in the nonsmoking patients (25). reviewing the field changes and their relationship with the most In another publication on chromosomal aberrations in important epidemiological risk factors as far as available data HNSCC patients, the investigators used microsatellite analysis justify. After evaluating whether these data are in support of (26). Allelic loss of chromosome 13 was detected in 10 of 16 either the migration or the independent origin theory, we will informative TAM samples when they were compared to blood pay attention to the studies in which clonal relationship between samples. No data on a relationship of this finding with smoking MPTs was studied and discuss whether data on clonality of could be detected. MPTs support any of both theories: migration of precursor cells Alterations in Cytokeratin Expression. Cytokeratins are the or independent tumor development. Finally, some clinical im- intermediate filament proteins found in the cytoplasm of all plications of these studies will be mentioned. epithelial cells. There are at least 20 different keratin polypep- tides that are expressed in different combinations, depending on Oral Field Changes and Their Relationship with Risk the type of epithelium and the degree of differentiation (27). Factors Aberrant expression of cytokeratins has been shown during the Morphological Changes. In 1962, Nieburgs et al. (15) re- process of HNSCC carcinogenesis (28, 29). Presence of cytok- ported malignancy-associated changes within smear cells of eratins 7, 8, 13, 16, and 19 was observed at abnormal anatom- normal buccal mucosa in patients with malignant disease. The ical sites or at abnormal intraepithelial levels in normal mucosa changes consisted of an increase in nuclear size, discontinuous from HNSCC patients (30–33). nuclear membrane, numerous Feulgen-negative areas, in- Only one study is available in which cytokeratin expres- creased associated chromatin surrounding the clear areas, and sion was studied in relation to smoking habits. Expression of

cytokeratins 7 and 8 in TAM occurred more frequently in the TAM compared with mRNA levels in control normal mucosa smoking group of patients than in the nonsmoking group (31). (40), but whether this relates to smoking is unknown. Changes in Blood Group Antigens of the ABH System. Increased Proliferation. One of the characteristics of a tumor Histo-blood group antigens are cell surface carbohydrates that is an increased proliferation. Shin et al. (47) showed a sequen- show changes in expression related to tissue type, differentia- tial increase in proliferating cell nuclear antigen expression in tion state and cell motility capacity. Type 2 chain ABH-carbo- head and neck tumorigenesis. hydrate structures are distributed broadly in epithelial and en- An increased number of proliferating epithelial cells was dothelial cells, independent of the patient’s ABO blood group. also shown in TAM from HNSCC patients (47, 48). This In normal oral and laryngeal epithelium, type 2 chain ABH- increase in proliferation was related rather to smoking than to antigens are expressed on parabasal cells (34). A 4-fold lower the presence of a HNSCC, as it was only detected in the TAM expression of type 2 chain ABH-antigen was shown in exfoli- from smoking HNSCC patients. No increase was observed in ated cells from macroscopically normal mucosa from six dif- TAM from nonsmoking HNSCC patients (49). This increase in ferent places distant from the HNSCC, compared with healthy proliferating cells was observed not only in TAM from smoking individuals (32). Because the ABH type 2 chain expression was HNSCC patients but also in mucosa of the UADT from healthy always lower in the mucosa from the patients than in the smokers (49). A trend toward an increased proliferation was mucosa from healthy controls, this antigen may be promising as also shown in the mucosa from ex-smoking cancer-free and a negative marker for field change and risk indication. HNSCC patients. Thus, the elevated proliferation persists for Foci of Cyclin D1 Expression. Cyclins are cell cycle regula- some time after quitting smoking. The higher proliferative tors that are functional only when associated with CDKs. Cy- activity in the epithelium of the UADT from smokers and clin D1 regulates the G1-S transition in the cell cycle and is ex-smokers may well constitute a fertile soil for genetic events functional when it is associated with either cdk4 or cdk6 (35). culminating in the development of HNSCC. Amplification of the chromosome 11q13 region, which results p53 Overexpression. Loss of function of the tumor suppressor in overexpression of the proto-oncogene cyclin D1 has been p53 can result in uncontrolled cell division and progressive described in about half of the HNSCC (36, 37). Cyclin D1 genomic instability (50). Abnormalities of the p53 tumor sup- amplification has been shown in premalignant lesions and the pressor gene are among the most frequent molecular events amplification frequency progresses from premalignant lesions in cancer. More than 90% of the HNSCCs contain mutated to invasive carcinoma (36). Bartkova et al. (37) observed p53 (51), and in 50% of the tumors, LOH of p53 has been clearly defined foci of cyclin D1 expression in sections of shown (52). normal mucosa adjacent to HNSCC that were not seen in Mutant p53 has a higher stability than wild-type p53, sections of normal mucosa from healthy individuals. Whether which allows accumulation to levels detectable by immunohis- this finding relates to smoking habits has not been addressed. tochemistry (53, 54). The frequency of p53-positive cells grad- Increased Expression of the Epidermal Growth Factor Re- ually increases as oral epithelium progresses from normal to ceptor. One of the cellular oncogenes that play a role in the hyperplasia to dysplasia to carcinoma (55, 56). Focal p53 development of HNSCC is the EGFR. This gene encodes the positivity was detected more often in normal TAM than in receptor of the growth factors epidermal growth factor and healthy control epithelium (56–60). Mutations in the p53 gene TGF-␣. Ligand binding to the extracellular domain of the were identified in both normal TAM and tumor-distant mucosa EGFR causes receptor dimerization, which activates tyrosine from HNSCC patients, in contrast to healthy nonsmokers (58, kinase function. This leads to autophosphorylation and subse- 61, 62). These mutations were polyclonal and differed from quent phosphorylation of intracellular target proteins, which those detected in the adjacent tumor (58, 61). results in proliferation (38). Focal overexpression of p53 might reflect an increased EGFR mRNA overexpression, as well as protein overex- risk of second primary tumors in these patients. Previously, it pression, has been demonstrated in nearly all HNSCCs (39– was shown that more p53 positive cell clusters were present in 41). EGFR expression increases during the development of epithelium surrounding multicentric HNSCC as compared to normal epithelium to dysplastic epithelium and further in- unicentric HNSCC (63). Furthermore, p53 expression above creases when dysplasia becomes an invasive carcinoma (42). the basal cell layers in oral mucosa has been found to be an Several studies have shown increased expression of the early event of malignant transformation and has predictive EGFR in TAM (39–46). In five of these studies, an overex- value for the development of HNSCC (60). This notion is in pression of the protein was observed (42–46), one study de- contrast with another study in which the investigators found scribed an elevated mRNA level (40), and one study showed that p53 overexpression in TAM could not predict the likeli- amplification of the EGFR gene (45). The EGFR overexpres- hood of a second primary squamous cell carcinoma (64). How- sion was almost as high in TAM from nonsmoking/nondrinking ever, no distinction was made between basal and suprabasal HNSCC patients as in TAM from smoking/drinking HNSCC p53 expression in that study. patients (43). No increase in EGFR expression was observed in The possible role of smoking in inducing p53 overexpres- mucosa from cancer-free smokers compared to cancer-free sion in TAM was examined by immunohistochemistry (57). No nonsmokers (43). Furthermore, both Grandis et al. (46) and differences in uniformly spread overexpression of p53 were Van Oijen et al. (43) showed that EGFR expression in the observed between the mucosa from smoking and nonsmoking mucosa from the HNSCC patients was less elevated when the HNSCC patients compared to healthy controls. epithelium was located more distant to the tumor (43, 46). In contrast to the uniformly spread p53 overexpression, These results suggest a paracrine effect on the EGFR expres- focal overexpression of p53 occurred more frequently in normal sion due to factors released by the tumor and not due to epithelium from smoking HNSCC patients than from nonsmok- influence of smoking. ing HNSCC patients and from healthy control individuals (57). Elevated TGF-␣ mRNA. Besides investigation of the EGFR However, the elevated number of p53 positive cell clusters also one of its ligands, TGF-␣, was investigated. It was shown could not be detected in mucosa from healthy smokers. This that the mRNA level of TGF-␣ was 5-fold increased in normal indicates that besides the exposure to tobacco, other environ-

mental factors and/or genetic factors must contribute to the growth and proliferation. A key participant in regulation of abundance of p53 positive cell clusters in TAM. Exposure to translation is the proto-oncogene product eIF4E or mRNA 5Ј alcohol was an additional factor in the HNSCC patients (57) cap-binding protein (72). This protein has been found to be and might have played a role in the development of the p53 expressed at an elevated level in HNSCC (73). Also, histolog- positive cell clusters together with the influence of tobacco. In ically normal margins of resected HNSCCs showed overex- addition to the findings in tissue sections (57), p53 overexpres- pression of eIF4E (73). Relationships with smoking habits were sion and mutations were detected in some clusters of cultured not investigated. cells from normal mucosa of healthy smokers (62). These data Protein Tyrosine Kinase and Protein Tyrosine Phosphatase on p53 gene alterations and p53 protein overexpression in Activity. Phosphorylation of proteins on tyrosyl residues is a apparently healthy TAM suggest a pivotal role for p53 in the key mechanism in signal transduction pathways that control early phase of oral carcinogenesis. growth, differentiation, and cellular architecture of normal and Lack of bcl-2 Expression. In normal tissue, there is a balance malignant cells (74). This phosphorylation is strictly regulated between cell proliferation and programmed cell death (apopto- by protein tyrosine kinases and protein tyrosine phosphatases. sis). Alterations of both pathways contribute to a clonal expan- Normal TAM showed a 2.2-fold increase in protein tyrosine sion of cancer cells. bcl-2, an apoptosis inhibitor, and its family kinase activity compared to the control mucosa from healthy members (among others, bax, an apoptosis inducer) play an individuals. In addition, in the TAM, a 1.7-fold elevated ratio of important role in the regulation of the apoptotic pathway (65). protein tyrosine kinase activity to protein tyrosine phosphatase Apoptosis itself did not vary significantly in the different stages activity was observed (75). Whether these findings are related of HNSCC tumorigenesis (66). However, there was lack of with smoking habits is unknown. bcl-2 expression in HNSCC and in normal TAM compared to A Relationship with Tobacco Exposure Has Been Estab- control mucosa. No data on relationship with smoking were lished for Some TAM Changes. When these data are sum- mentioned (66). Because bcl-2 is supposed to inhibit apoptosis, marized, it appears that TAM contains a lot of alterations, one would expect an increase in bcl-2 expression during tu- which means that genetic abnormalities are indeed distributed morigenesis and therefore the lack of bcl-2 expression is rather throughout the epithelium of the entire UADT. Additional surprising. However, to estimate the bcl-2 activity, the expres- research is required to assess which field changes have carci- sion of bcl-2 has to be interpreted in the context of levels of nogenetic significance and which are merely epiphenomenal. other bcl-2/bax family members. It seems that TAM from smoking HNSCC patients shows Increased Glutathione S-Transferase. Glutathione S-trans- alterations not found in nonsmoking patients (Fig. 2), which ferases are detoxification enzymes that comprise different iso- implies that TAM in smoking patients harbors altered cells that classes (␣-, ␮-, ␲-, and ␶-class), of which the expression levels are absent in nonsmoking patients. If these cells have migrated are tissue specific and dependent on polymorphisms (67). Glu- from adjacent tumors, one would expect some changes in TAM tathione S-transferases are multifunctional, intracellular, solu- from nonsmoking patients, which is not supported by these ble, or membrane-bound enzymes, which catalyze the conju- data. In addition, some of the field changes observed (increased gation of many electrophilic hydrophobic compounds with the nuclear area, increased proliferation, and mutations in p53) tripeptide GSH. Glutathione S-transferase ␮ is an isozyme with were already present in healthy smokers without a tumor being a marked specificity for catalyzing the conjugation of epoxides, present as source of migrating cells (19, 49, 62). Therefore, such as benzo(a)-4,5-oxide and sterene-7–8-oxide, carcino- unless we assume that TAM in both smoking and nonsmoking genic components in cigarette smoke (68). A 2-fold higher risk patients contains migrating cells that do not betray themselves of laryngeal cancer has been shown among smokers who lack by showing as yet unknown alterations, these results support the glutathione S-transferase ␮ isoenzyme (69). In contrast, the idea that TAM changes in HNSCC patients are carcinogen glutathione S-transferases were increased in dysplastic oral induced, independent events rather than being the result of lesions and HNSCC (70). migrated transformed cells. The expression of all glutathione S-transferase isoenzymes This notion is also supported by the epidemiological find- was significantly higher in the suprabasal and superficial layers ing that the risk of developing second primary HNSCCs is of normal oral mucosa from HNSCC patients who subsequently higher in smokers/drinkers than in nonsmokers/nondrinkers (8, developed a second primary tumor than in normal oral mucosa 9). Moreover, this risk decreases when the patient quits smok- from HNSCC patients who were free of disease for at least 7 ing and stops abusing alcohol (8). years (71). Also, in cell scrapes of macroscopically normal TAM, elevated levels of glutathione S-transferase ␮- and Clonality Studies ␲-class were observed (71). Whether these findings have any As outlined in the Introduction, comparing the genetic alter- relationship with smoking habits has not been assessed. The ations occurring in MPTs of the head and neck area will also be reason for these increased levels is an intriguing fact, because helpful in assessing the strength of either the migration or the elevated levels of detoxification enzymes protect against car- independency theory in explaining oral field cancerization. If cinogenic attacks. It may reflect a futile response to the pres- multiple tumors develop due to migration of malignant cells ence of carcinogenic metabolites from tobacco, because all from a primary source, then the tumors and dysplasias from the investigated individuals smoked. It becomes even more com- same patient should show identical genetic alterations, whereas plex when one realizes that malignant tissue might benefit from in case of independent origin, these alterations will be different. the higher levels of glutathione S-transferase in case of chem- For these studies, various clonal markers have been used. otherapy (70). The reason for the high levels of glutathione Clonal Markers. To investigate the relationship between S-transferase may not yet be clear; nevertheless, it seems to MPTs, good clonal markers are needed. To qualify as a marker, have a predictive value for the development of a (second) such a genetic alteration should (a) occur very early in the primary tumor. development of the primary lesion, (b) be maintained during Expression of the Proto-oncogene Product eIF4E. Control progression of the lesion, (c) exhibit sufficient variability, and of gene expression at the translational level is important in cell (d) be applicable in the majority of the lesions. We will mention

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

the genetic markers that have been used to assess the clonal evidenced by showing identical microsatellite alterations, LOH relationship between separate squamous lesions in the aerodi- patterns, or cytogenetic features (14, 78, 79, 90). However, the gestive tract and discuss whether they meet the above outlined overwhelming majority of remote MPTs show no clonal rela- 4 requirements. tionships and can therefore be assumed to have developed One of the genetic alterations used is the pattern of X- independently. chromosome inactivation (13). However, when using X-chro- HNSCC or adjacent premalignant lesions that are located mosome inactivation as marker for investigation of the clonal very close to each other more often show identical genetic relation between tumors lying close to each other, one has to changes. Califano et al. (12) observed a clonal relationship realize that X-chromosome inactivation occurs relatively early between five HNSCCs and the tumor-adjacent premaligant in embryonic development, resulting in large patches of cells lesions by using LOH pattern analysis. Two patients with derived from a common ancestor cell and thus having the same synchronous HNSCC tumors lying close to each other were X-chromosome inactivated (76). In addition, X-chromosome investigated for clonality by Van Oijen et al. (80). The first inactivation can be nonrandom or cell type specific (77). patient showed an identical p53 mutation and an identical LOH Karyotypes of tumors have also been used as a clonal pattern in both tumors, whereas the other patient did not show marker (78). However, tissue culture is required for this type of identical aberrations. This strongly suggests that in the first analysis, which might cause additional genetic alterations. patient, migration of malignant cells has occurred. In the nine Another kind of clonal markers that has been used in- other patients investigated with HNSCC lying remote from volves LOH patterns for microsatellite markers at different each other, no clonality was observed. Thus, in patients in chromosomal loci (12, 14, 79, 80). LOH at loci on chromosome whom two HNSCCs are not separated by a large distance, some arms 3p, 9p, and 17p have been shown to occur early in tumors might have developed from the same primary lesion, carcinogenesis (12, 80–84). Only a single change might occur whereas other tumors have developed independently. in a LOH pattern of about 10 microsatellite markers during More evidence for the existence of lesions arising inde- tumor progression and metastasizing (80, 85). However, LOH pendently in the UADT came from the diversity of p53 muta- at loci on chromosomes 3p, 9p, and 17p occurs so frequently tions identified in histologically normal TAM from HNSCC (80) that complete loss of all markers may be present in many patients (58, 61). All identified mutations differed from the p53 of the lesions, which means that the variability of these markers mutation in the matched HNSCC. This excludes the possibility is not high enough to draw conclusions about clonal relation- that new lesions in these patients develop by migrated tumor ships between all lesions. cells, as well as the explanation that early lesions in TAM occur Finally, p53 mutations have been used as a clonal marker due to migration of progenitor cells with a p53 mutation. (51, 55, 61, 80, 86). p53 mutations are an early event in the One precaution needs to be considered. The conclusion development of HNSCC because they are already present in that MPTs develop independently is only tenable when one can normal tissue distant from tumors (56, 58, 61), in normal tissue exclude the possibility that there is migration of cells with a yet from healthy smokers (62), and in premalignant lesions (56). unknown genetic alteration that had arisen prior to all “early These mutations are diverse enough (80) and stable during alterations” investigated. However, it is difficult to imagine that metastasizing (87). Thus p53 mutations in HNSCC patients progenitor cells, with only a minimal genetic alteration, would appear to be very useful as clonal marker, whereas the other have the capacity to migrate intraepithelially over large dis- markers are less suitable due to lack of variability and stability tances. Neither is it very plausible that such progenitor cells or due to technical requirements. would be displaced by salivary flow, after which they settle Polyclonality of MPTs in the Head and Neck. Most studies elsewhere for outgrowth, because progenitor cells do not have that used clonal markers to investigate the relationship between any invasive or metastatic potential. Furthermore, it has been MPTs or to investigate dysplastic lesions occurring in the shown that a few “early” transformed cells that are surrounded UADT and that were remote from each other showed poly- by normal cells do not succeed in the development of a new clonality between these lesions (14, 79, 80, 86, 88, 89). Only a lesion (91). limited amount of MPTs showed the same genetic alterations as In the bronchial tree, the situation may be different, as

Franklin et al. (92) reported a case harboring the same p53 14. Partridge, M., Emilion, G., Pateromichelakis, S., Phillips, E., and Langdon, mutation in dysplastic bronchial mucosal samples taken from J. Field Cancerisation of the oral cavity: comparison of the spectrum of molecular alterations in cases presenting with both dysplastic and malignant lesions. Oral different locations. Migration of p53 mutated progenitor cells is Oncol., 33: 332–337, 1997. highly likely to have occurred in that patient, unless this par- 15. Nieburgs, H. E., Herman, B. E., and Reisman, H. Buccal cell changes in ticular p53 mutation was induced independently in these dif- patients with malignant tumours. Lab. Invest., 11: 80–88, 1962. ferent lesions as a consequence of a specific carcinogen. 16. Incze, J., Vaughan, C. W., Lui, P., Strong, M. S., and Kulapaditharom, B. Premalignant changes in normal appearing epithelium in patients with squamous cell carcinoma of the upper aerodigestive tract. Am. J. Surg., 144: 401–405, 1982. Clinical Relevance and Conclusions 17. Ogden, G. R., Cowpe, J. G., and Green, M. W. The effect of distant The finding that field changes frequently occur in TAM of malignancy upon quantitative cytologic assessment of normal oral mucosa. Can- HNSCC patients creates a different view on tumor excision cer (Phila.), 65: 477–480, 1990. margins that contain molecularly altered cells (93, 94). The 18. Ogden, G. R., Cowpe, J. G., and Green, M. W. Detection of field change in conclusion that the margin is tumor positive if it shows the oral cancer using oral exfoliative cytologic study. Cancer (Phila.), 68: 1611–1615, presence of genetically altered cells does not necessarily hold 1991. true for every molecular marker used. The molecular marker 19. Ogden, G. R., Cowpe, J. G., and Green, M. W. Quantitative exfoliative cytology of normal buccal mucosa: effect of smoking. J. Oral Pathol. Med., 19: used has to be specific for that particular tumor. 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Monique G. C. T. van Oijen and Pieter J. Slootweg

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