Angela Flavia Logullo, Luiz Paulo Kowalski, Sueli Nonogaki,Roberto E. \I Miguel, Humberto Torloni, Ricardo R. 8rentani

P53 overexpression in epidermoid carcinomaof the head and neck

Ludwig lnstitute for Cancer ResearchDepart111ent of Head and Neck Surgery, A.C. Camargo Hospital, Fundação Antonio Prudente - São Paulo, Brazil

The theory of field cancerization in tumors of the head and neck reflects the complex oncogenesis that occurs in this region. Themechanisms that control cell proliferation at the molecular levei in epidermoid carcinomas (ECs) of the upper aerodigestive tract arestill unclear. Mutations in p53 are the genetic alterations most often detected in ECs of the head and neck and seem to contributeactively to the carcinogenic process triggered by p53 as a tumor-suppressor gene and to its association with tobacco. The objective ofthe present study was to investigate the expression of p53 protein in epidermoid head and neck carcinomas by immunohistochemistryand its immunohistochemical correlation with other prognostic factors. The study was conducted on 63 consecutive ECs cases notsubmitted to previous treatment. Specimens of the tumor and of the normal adjacent mucosa were collected during surgery andsubmitted to immunohistochemical reaction for the determination of the expression of anti-protein p53 antibody (M7001 DAKO AIS,Denmark). Anatomo-c1inical and demographic data were not significantly correlated with the presence of Iymph node metastases orp53 expression in the tumor or in the adjacent normal mucosa. Tumor localization in the larynx was significantly correlated with p53expression. Histological grading as grades I, 11,111and IV was correlated with significant p53 expression (p = 0.025). Conclusions: 1)in the studied material obtained from 63 cases of head and neck ECs, we detected a 48 percent rate of immunohistochemicallydetectable p53 overexpression; 2) we did not detect a relationship between demographic patient data and p53 expression in the tumoror in the normal adjacent mucosa; 3) p53 overexpression was significantly more frequent in ECs material from the larynx; and 4) Thepresence of 12 cases with p53 overexpression in the normal adjacent mucosa and with a p53-negative tumor is in agreement with thetheory of field cancerization. Follow-up of this patient series for a longe r period of time will permit a better analysis of these values.

UNITERMS: P53, squamous cell carcinoma, head and neck cancer, immunohistochemistry

INTRODUCTION

EPidermoid carcinomas (ECs) represent more than90 percent of the neoplasias of the upperaerodigestive trace) and their incidence increases

among subjects who smoke cigarettes and consumealcoholic beverages.12 ECs correspond to the sixth mostfrequent form of cancer in the worldY

Address for correspondence:Angela Flavia LogulloRua Prot. Antonio Prudente, 211São Paulo/SP - Brasil- CEP 01509-010

Patients with head and neck ECs in the sanle stageITIayrespond differently to different forms of treatlnent.2

Currently recognized prognostic factors are not sufficientlyobjective or standardized, and do not correspond to thebiological behavior of the tumor at the cellular leveI.

The theory of field cancerization in head and neckneoplasias reflects the complex oncogenesis that occursin this region.33 The frequent occurrence of ITIultipleprimary tumors in the upper aerodigestive tract Inay beexplained by continuous exposure of the ITIUCOSato variollscarcinogens. However, the sequential stages of thiscomplex carcinogenesis have not been flllly elucidated andthe molecular mechanisms that control cell proliferationand the aberrant behaviorofECs in the upper aerodigestivetract are still unknown.

LOGULLO, A.F.; KOWALSKI, L.P.; NONOGAKI, S. e aI. - P53 overexpression in epidermoidcarcinoma of the head and neck

São Paulo Medicai Journal/RPM 115(1): 1349-1355, 1997

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lmmunohistochemistry

MA-TERIAL AND METHODS

The study was conducted on 63 consecutive ECscases not submitted to previous treatment registered atthe Department of Head and Neck Surgery of the A.C.Camargo Hospital in 1994 and 1995. Tumor and nonn~ladjacent mucosa specimens were collected during surgeryand fixed in Carnoy solution. After fixation, the materialwas embedded in paraffin and 4 ~m thick sections wereobtained and stained with HE. The material was thensubmitted to immunohistochemical reaction for thedetermination of the expression of anti-protein p53antibody (anti-human p53 protein clone D07, M700 IDAKO, AIS, Denmark).

p=0.424

p=0.45

p=0.485

p=0.89

p=o.51

p=1.00

819

512

15

239

25

The material was deparaffinized and immersed in abath containing decreasing concentrations of xylol andethyl alcohol. Tne antigen was recovered by treatment ina microwave oven under immersion in a nitric citratesolution, with 4 baths of 5 min each at lnaxilnum potency.Endogenous peroxidase (H202) was blocked and thematerial was incubated 1: 100 with primary antibodyovernight at 4°C, followed by incubation with I:200biotinylated secondary antibody for 30 lnin at 37°C. Afterincubation with 1:800 StrepAB complex:HRP for 30 lnin

at 37°C (DAKO StrepABcOlnplex/GRP Duet,mouse/rabbit kit), thematerial was ilnmersed in60 mg percent DAB (2,3diaminobenzidine tetra-hydrochloride, SiglnaChemical Co., D5637) plus1 percent dilnethyl sulfo-xide PA (Labsynth) for 5min at 37°C and coun-terstained with helna-toxylin.

When more than 20percent of the tumor cellnuclei stained dark brown,the sample was consideredpositive. The same criterionwas used for the evaluationof normal adjacent mucosa. _Positive and negativecontrols were systelna-tically performed for eachreaction.

413

13

246

26

2

622

p53 expression

(+) (-)

26 254 7

28 30

1 O1 1

present

absent

present

absent

present

absent

Categories

male

female

White

Asian

Black

< 45

46-60

>60

Table 1Correlation between p53 expression and demographic data

Age

Family

History

Smoking

habit

alcoholism

Race

Variables

Sex

Genes involved in carcinogenesis may be classifiedas proto-oncogenes and tumor-suppressor genes accordingto their action mechanism.23,35 Tumor-suppressor genesco de for proteins with functions related to the control andsuppression of cell growth and proliferation bytransactivation or transrepression of specific genes. Thesegenes actively affect the cell cycle, regulating it in arepressi ve manner. II

The role of gene p53 has been the subject of severalstudies on solid human tumors.5,6,15,16,IX,19,25,26,30P53mutations are the genetic alterations most frequentlydetected in head and neck ECS,2XJ4specifically in thelarynx, and seem to actively contribute to the carcinogenicprocess of the gene31 as a tumor suppressor and to itsassociation with tobacco.4,IO,17

The accumulation of lnutation of gene p53 associatedwith head and neck ECs has been reported in the literature,although it has not been correlated with other prognosticfactors.1.2,7.13 A better understanding of the role andmechanism of action of gene p53 in the various stages ofcarcinogenesis may be of help in terms of specifictherapeutic approaches, and may identify new tumormarkers for early diagnosis.

The objective of the present study was to investigatethe expression of p53 protein in head anCl neck ECs byilnmunohistochemistry and to determine its relation toother prognostic factors.

São Paulo Medicai JournallRPM 115(1): 1349-1355, 1997 LOGULLO, A.F.; KOWALSKI, L.P.; NONOGAKI, S. et aI. - P53 overexpression in epidermoidcarcinoma of the head and neck

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Figure 1 - p53 expression in grade II epidermoid carcinoma of the tongue.

Statistica/ ana/ysis

Double-entry convergence tables were constructedfor the analysis of correlations between the clinical-pathologic variables studied and the immunohistochemicalexpression of mutated p53 protein. Statistical significancewas calculated by the chi -square test or by the exact Fishertest when applicable.

RESULTS

Expression of mutated p53 protein was consideredto be posi tive in 30 of the 63 specimens of primary tumors(48 percent). The study did not show a significantcorrelation between protein p53 overexpression and patientdemographic variables (family history of cancer, cigarettesmoking or alcohol consumption) (Table 1). Cases for

whom demographic data were not available were excludedfrom the table.

Figure 1 shows a grade 11EC of the tongue with p53overexpression.

To facilitate data analysis, the tumors wereschematically assigned to the three following topographies:pharynx, including the cervical esophagus; oral cavity,including palate, mouth fIoor, tongue and gingiva; andlarynx. Table 2 shows the correlation between p53expression and anatomical location, histologic grade andlymph node status.

Epidermoid carcinoma of the larynx was significantlycorrelated with p53 expression which did not occur in otherlocations.

Histological grading was schematically divided intogrades 11. 111 and IV. Usually, grades 111 and IV have thebest prognosis. Correlation with p53 expression wassignificant (p = 0.025). No correlation was observedbetween p53 expression and presence of lymph nodemetastases.

LOGULLO, A.F.; KOWALSKI, L.P.; NONOGAKI, S. et aI. - P53 overexpression in epidermoidcarcinoma of the head and neck

São Paulo Medicai Journal/RPM 115(1): 1349-1355, 1997

1352

p=0.20

p=0.025

284

14

19

27

3

17

13

negative

positive

DISCUSSION

The hypothesis of the participation of gene p53 inthe process of carcinogenesis of head and neck tumorshas been discussed in severaI studies.7-lJ.'3.'4.21.24.27.2lJ,32.34.37After the discovery of anti-protein p53 antibodies, severaIretrospective studies have been published, with variableresults, i.e., rates of p53 positivity of 30 to 67 percent.Thirty to 65 percent of the carcinomas have characteristicl11utations in gene p53, with an allele usually having a pointInutation and producing a mis-sense p53 protein thataccumulates at high leveIs in cancer cells. The second allelewas Iost by conversion and/or genetic deletion.22

Celllines from ECs of the upper aerodigestive tracthave revealed increased p53 expression in vitro.1.3ó

Data concerning p53 expression in the normaladjacent l11ucosa and in the tumor of the same patient arepresented in Table 3. The results show that p53 expressionat these two sites was not coincident in 29 cases. The tumorwas p53+ and the lnucosa p53- in 17 cases, and 12 casespresented opposite results (a p53- tumor and a p53+adjacent lnucosa).

Figure 2 shows an area ofthe nOrInal adjacent mucosawith lnoderate epithelial dysplasia and strongly p53positive. Figure 3 shows an area of non11al adjacent l11ucosathat is negative for p53 overexpression.

Although the follow-up til11eof the present series isstill short, three patients who presented recurrences hadp53+ tUl11ors, whereas only one case had a recurrenceamong patients without p53 expression. This sl11allnUlnberof recurrences prevents statisticaI analysis.

Tumor topography

Oral cavity

Pharynx

Larynx

Histological grade

I + II111 + IVLymph node

metastases

Mutations, deletions anelTable 2

Association between p53 expression and tumor topography, histological grade al11plification ofthe undeleteeland Iymph node involvement allele of gene p53 are

--------------------------------- COl11111onIy fou neI in thesep53 expression p=0.0225 lines.:n

(+) (-) Co nside r in g the 111u 1-15 18 ticentric etiology of tun10rs of6 12 the aerodigestive tract,10 2 several investigators have

a Iso e x a111ine d the no rn1a Iprelesional adjacent ll1ucosaof some cases and havedetected an irregular patternof p53 expression, althoughthey elid not detect acorrelation between p53

expression in the non11al mucosa adjacent to the turnorand the appearance of a second neoplasia orrecurrence.14.20.27.2lJ,32

Brachlnan et aI.2 investigated 31 specir11ens of headand neck ECs and observed rnutations of gene p53 in 53percent of cases, demonstrated by polYlnorphisll1 of singlestrand confon11ation. The salne investigators later detecteelmutations in gene p53 by the polYlnerase chain re£1ctionin 63 percent of the cell lines studied. Of the 3 I cases ofhead and neck ECs studied by Chung et £11.,72 I presenteeiat least one mutation of gene p53, analyzed by the singlechain polymorphisl11 confon11ation l11ethod. Field et aI.,1Oin a study of73 ECs cases, observed the presence of proteinp 5 3 o ver e xp re s s io n in 67 p e rc e n t o f t he In b yimmunohistochemistry. In another il11l11unohistochelnicalstudy, Frank et aI.I3 detected p53 overexpression in 37percent of cases of ECs of the hypopharynx. Shin et £11.32obtained a 43 percent index in an il11111unohistocheI11ic£11study of head and neck ECs. The presence of p53+ in 48percent of the 63 head and neck ECs cases positive forp53 in our study is compatible with literature data.

In vitro studies carried out by Yin et aI.,37S0l11erSetaI. 34and Weinberg et aI. 3óshowed abnorI11alities of genep53 in celllines froln head and neck carcinolnas, such asoverexpression of protein p53 del110nstrated byil11111unohistochel11istryand mutations and deletions of thegene detected by sequential analysis by the polYlnerasechain reaction.

Maestro et aI.24investigated 58 cases of larynx tunlorsand found p53 overexpression in 60 percent using £1niml11unohistochemicallnethod and l110lecular analysis bythe polymerase chain reaction and in situ hybridization.The authors concluded that this is the genetic £1lterationmost frequently occurring in these tUI11ors.Of 89 cases ofECs of the larynx studied by NadaI et aI.,27 64 percentpresented il11munohistochelnically-d~tecteel p53

São Paulo Medicai Journal/RPM 115(1): 1349-1355, 1997 LOGULLO, A.F.; KOWALSKI, L.P.; NONOGAKI, S. et aI. - P53 overexpression in epidermoidcarcinoma of the head and neck

1353

Figure 2 - Normal adjacent mucosa with moderate epithelial dysplasia and strongly p53 positive.

overexpression. The specific localization for these case.sof cancer of the larynx was statisticalIy significant in termsof p53 expression, a fact not previously reported.

The p53 overexpression observed in 10 of the 12cases of cancer of the larynx studied here was morefrequent than in the study discussed above. Nees et aI.(1993) found p53 overexpression in only 44 percent oftheir cases of ECs of the larynx and in 80 percent of thecases of cancer of the oropharynx. We are currentlyexpanding our study to determine p53 expression in thevarious topographies of the head and neck.

Prospective studies with appropriate material foranalysis of p53 have only permitted a simple statisticalcorrelation with some isolated factors such as smoking4.IOand hUlTIanpapilIomavirus.3 The absence of a correlationbetween smoking and p53 gene expression in the presentstudy disagrees with the data reported by others.2,4

With a longer foIlow-up time, few studies wereconsistent in showing the absence of a correlation betweenthe prognostic factors normalIy used and p53 expression

in head and neck tumors. Frank et aI.I3 did not detect acorrelation between p53 expression in ECs of thehypopharynx and other data such as incidence of secondarytumors, survival, ploidy or histological grade. NadaI etaI.27 did not find a prognostic significance of p53expression in neoplastic and dysplastic lesions of the Iarynxin a study of 89 cases, and concluded that the presence oflTIutations of gene p53 detected by the poIYlTIeraSe chainreaction is not correlated with radiosensitivity in thesetumors.

Taking into account the theory of field cancerization,Nees et aI.29 identified mutations of gene p53 in tUlTIOrSand in the normal adjacent mucosa and suggested theparticipation of p53 in the carcinogenesis of lTIultipIetLllTIOrs. Shin et aI.32 and Gusterson et aI.I4 studiedpremalignant lesions of the squamous epitheIiulTI,including dysplasia, hyperplasia, Bowen disease, and thenormal adjacent mucosa and found 21 to 45 percent and15 to 16.6 percent p53 positivity by immunochemistry intheir respective series. The scarcity of data concerning

LOGULLO, A.F.; KOWALSKI, L.P.; NONOGAKI, S. et aI. - P53 overexpression in epidermoidcarcinoma of the head and neck

São Paulo Medicai Journal/RPM 115(1): 1349-1355, 1997

1354

survival prevented these authors from correlating theirresults with prognostic factors. However, there is noobjecti ve evidence of a correlation between p53 expressionand prognostic factors for head and neck carcinomas.27

The absence of a correlation between the expressionof p53 in the normal adjacent lTIUCOSaand in the tumor areaof the same patients detected here agrees with literature data.Nees et £11.29 found p53 overexpression in the normaladjacent mucosa and absence of p53 protein in patients withhead and neck ECs This apparent discrepancy was observedin 12 of our cases. The presence of p53 expression in theadjacent mucosa may indicate the possible origin of multipletumors or the local recurrence in this area through differentcarcinogenic processes, involving or not mutation of thep53 gene. The specific localization in cancer of the larynxwas statistically significant in terms of p53 expression, afact that is not uncommon in literature reports. Our seriesmust be expanded for a better analysis of the topographicdistribution of tumors according to p53 expression.

Table 3Expression of p53 in the primary tumor and in the

adjacent mucosa.

CONCLUSIONS

1. In material obtained from 63 cases of head and neckECs, we observed 48 percent imlnunohistochelTIically-detectable p53 overexpression, a rate compatible withliterature data.

2. We did not observe a relation between patientdemographic data and p53 expression in the tUlnor orin the normal adjacent mucosa.

3. p53 overexpression was significantly more frequentin material from ECs of the larynx.

4. The presence of 12 cases with p53 overexpression inthe normal adjacent mucosa and a p53-negative tUlnoragrees with the theory of field cancerization. Thisfinding may be a factor for the early detection ofpatients at high risk to develop other p53-positivetumors.A longer follow-up of this series \vill pennit a better

analysis of these values in terms of recurrence anel thepossible appearance of other tumors, which is aboLIt 6percent (unpublisheel data).

p53( +) expression p53( -) expression

in the tumor

p53(+)

expression

p53(-)

expression

13

17

in mucosa

12

20

ACKNOWLEDGMENTS

We wish to thank Myiuki Fukuela da Silva anel CarlosFerreira Nascimento for technical assistance. A.F.I. is therecipient of a Doctoral fellowship from FAPESP.

São Paulo Medicai JournallRPM 115(1): 1349-1355, 1997 LOGULLO, A.F.; KOWALSKI, L.P.; NONOGALI, S. et aI. - P53 overexpression in epidermoidcarcinoma of the head and neck

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LOGULLO, A.F.; KOWALSKI, L.P.; NONOGAKI, s. et aI. - P53 overexpression in epidermoidcarcinoma of the head and neck

São Paulo Medicai Journal/RPM 115(1): 1349-1355, 1997