Sao Paulo Med. J. vol.118 número3

INTRODUCTION

The large num b e r o f histo lo gical type s o f

tum o rs in salivary glands tum ake s the tum a ve ry he te ro

-ge ne o us gro up o f ne o plasias. The gre ate st

difficultie s in the ir ide ntificatio n are due to the m o rpho

-lo gical varie ty and co m ple xity that co m pro m ise the

histo p atho lo gic al d iagno sis and c lassific atio n o f

the se tum o rs.

1

Muco epidermo id carcino ma (MEC) represents

5 to 12% o f salivary gland tumo rs, with the mo st co

m-mo n site b eing the paro tid gland (70 to 90%).

2-7

_In

the o ral cavity, the palate is the mo st affected site

and the lip is the site with least incidence.

8

In additio n to the cyto lo gical and mo rpho lo

gi-cal pro pe rtie s o f the se ne o plasias, o ne o f the m o st

im po rtant crite ria fo r the m e asure m e nt o f the ir b io

-lo gical b e havio r and aggre ssive ne ss is ce ll pro

lif-e ratio n. In this way, im m uno histo chlif-e m ical m arklif-e rs

o f ce ll pro life ratio n have b e e n fo und to b e use ful in

tum o r c lassific atio n and have fo rm e d p art o f the

pro gno stic and therapeutic studies o f these patho lo

-gie s.

9,10

The pro life rating ce ll nucle ar antige n (PCNA)

ide ntifie d b y Myiach e t al

11

_{is a 36 kd, acidic, no}

n-histo ne nucle ar pro te in that he lps the de lta DNA

po lym e rase in DNA synthe sis.

12

_{This pro te in has a}

high co ncentratio n in the late G1 and early S phases,

dim inishe s in the G2 phase and is alm o st ab se nt in

the M phase .

12,13

The e valuatio n o f c e ll p ro life ratio n us ing

PCNA is co m parab le to and, unde r ce rtain co

ndi-tio ns, supe rio r to the tradindi-tio nal me tho ds o f mito tic

Original Article

REVISTA PAULISTA DE MEDICIN A

Prolife rating ce ll nucle ar antige n e xpre ssion in

mucoe pide rmoid carcinoma of salivary glands

Head and Neck Surgery Department of Heliópolis Hospital and the Pathology

Department of the Dentistry Faculty, São Paulo University, São Paulo, Brazil.

a b s t r a c t

CON TEX T:

Amo ng the cyto lo g ical and mo rpho lo g ical pro perties o f muco epidermo id carcino ma, o ne o f the mo st impo rtant criteria fo r measuring its bio lo g ical behavio r and ag g ressiveness is cell pro lif-eratio n. In this way, immuno histo chemical markers o f cell pro lifera-tio n have been fo und to be useful in tumo r classificalifera-tio n and have fo rmed part o f the pro g no stic and therapeutic studies o f these pa-tho lo g ies.

OBJECTIVE:

To analyz e 1 1 cases o f muco epidermo id carcino ma (MEC) using the pro liferatio n activity marker (PCN A) and to deter-mine its relatio nship to the g rade o f malig nancy o f these tumo rs.

DESIGN :

Co rrelatio n study.

SETTIN G:

Head and N eck Surg ery Service o f Helió po lis Ho spital, São Paulo , Braz il.

SAM PLE:

Slides o f 1 1 cases o f primary muco epidermo id carcino -mas o f salivary g lands were prepared acco rding to ro utine tech-niques emplo yed in the O ral Patho lo g y Department o f the Dentistry Faculty o f São Paulo University, Braz il. They were fixed in a 1 0 % fo rmaldehyde so lutio n and stained with hemato xylin and eo sin. Af-ter this preparatio n the tumo rs were classified as lo w, inAf-termediate and hig h g rade o f malig nancy, acco rding to the criteria established by Seifert & So bin and Auclair, G o o de & Ellis. The slides were sent fo r immuno histo chemical pro cessing to evaluate the po sitivity o f pro -liferating cell nuclear antig en using the streptavidin bio tin technique.

M AIN M EASUREM EN T:

The co rrelatio n between pro liferating cell nuclear antig en expressio n and the histo lo g ical malig nancy g rade in muco epidermo id carcino ma o f salivary g lands.

RESULTS:

there were 4 cases (3 6 %) o f lo w g rade, 4 cases (3 6 %) o f intermediate g rade and 3 cases (2 7 %) o f hig h g rade o f malig nancy. After a co mparative study between histo lo g ical features and immu-no histo chemical analysis, sig nificant differences were o bserved (P < 0 .0 1 ) fo r lo w, intermediate and hig h g rades: 1 6 .0 4 %, 2 6 .9 8 % and 5 6 .9 8 % o f pro liferating cell nuclear antig en expressio n in mu-co epidermo id carcino ma , respectively.

CON CLUSION :

The pro liferating cell nuclear antig en expressio n in-creases with the g rade o f malig nancy in muco epidermo id carcino ma o f salivary g lands.

KEY W ORDS:

Pro liferating cell nuclear antig en. Muco epidermo id Carcino ma. Salivary G land.

figure c o unt us ing o p tic al m ic ro s c o p y, tritiate d

tim idin uptake and flo w cyto m e try.

9,14-16

This study was pe rfo rm e d to ve rify the pre

s-e ncs-e o f a co rrs-e latio n b s-e tws-e s-e n PCNA s-e xprs-e ssio n and

the histo lo gical m alignancy grade in MEC o f

sali-vary glands.

METHODS

In a re tro spe ctive analysis o f the re co rds o f

the He ad and Ne ck Surge ry Se rvice o f He lió po lis

Ho spital, 25 case s o f MEC o f salivary glands we re

fo und b e twe e n 1978 and 1997. In e le ve n case s the

sam ple b lo cks

we re suitab le fo r re e valuatio n o f the

histo p atho lo gic al d iagno sis and fo r im m uno

his-to che m ical analysis.

The c linic al d ata o n the p atie nts (age , se x,

race , pre se nce o f sym pto m s and o utco m e ) and

tu-m o rs (site and size ) we re co llate d.

Diagnostic tests

a)

Conventional histopathological study. Fo r this

analy-sis, tissue se c tio ns with 3-4 m m thic kne ss, fixe d

in 10% fo rm ald e hyd e so lutio n and e m b e d d e d in

p araffin we re e xam ine d . The slid e s we re staine d

with he m ato xylin and e o sin and stud ie d using

o p tic al m ic ro sc o p y. The tum o rs we re grad e d fo r

m alignanc y, ac c o rd ing to the c rite ria o f Se ife rt

& Sab in

5,6

and Auclair, Go o de & Ellis,

17

as fo llo ws:

(a) Lo w grad e : highly d iffe re ntiate d ne o p lasia

with p re d o m inanc e o f m ac ro and m ic ro c ysts.

Pre se nc e o f inte rm e d iate and m uc in-p ro d uc ing

c e lls (

Figure 1);

(b ) Inte rm e d iate grad e : p re d o m

i-nanc e o f inte rm e d iate c e lls and fe w c ysts. Pre

s-e nc s-e o f m uc in-p ro d uc ing c s-e lls and island s o f

e p id e rm o id c e lls (

Figure 2

) ; ( c ) Hig h g rad e :

p o o rly d iffe re ntiate d ne o p lasia with p re d o m

i-nanc e o f inte rm e d iate and e p id e rm o id c e lls in

so lid b lo c ks. Muc in-p ro d uc ing c e lls are p re se nt

Figure1 -

Low malignancy grade MEC of a salivary gland. Note the

mucin cell with wide, clear cytoplasm and the nucleus displaced to the

periphery, lining the cystic spaces. (Stained with hematoxylin and eosin

with magnification of 250x).

in le ss than 10% (

Figure 3

).

b )

Immunohistochemical study for PCNA determination. Fo r

im m u n o h is to c h e m ic al PCNA d e te c tio n , th e

stre p tavid in b io tin te c hniq ue was use d .

15

The

mo no clo nal antib o dy use d was PC10 (DAKO Co

r-po ratio n, Glo strup, De nm ark) in a 1/100 dilutio n

with 18 ho urs incub atio n tim e . Tum o r ce lls that

sho we d a b ro wnish stain we re co nside re d po

si-tive (

Figure 4

). The q uantitative e xp re ssio n o f

PCNA was o b taine d fro m the re latio nship b e

-tween the numb er o f PCNA-po sitive cells and the

to tal num b e r o f e valuate d c e lls in p e rc e ntage

te rm s. The to tal num b e r o f e valuate d ce lls was

ne ve r le ss than 1000 ce lls in e ach pro ce dure . Two

e valuatio ns we re pe rfo rm e d fo r e ach tum o r. In

all case s the im m uno histo che m ical e valuatio n

o f PCNA was pe rfo rm e d b y the patho lo gist in a

b lind m anne r (witho ut kno wing the histo patho

-lo gical diagno sis o r the m alignancy grade ).

Main Measurement

The co rrelatio n b etween PCNA expressio n and

the histo lo gical grade o f m alignancy in MEC o f

sali-vary glands.

S tatistical methods

The statistical analysis was pe rfo rm e d using

the SAS/STAT pro gram, users’ guide versio n 6.0 (SAS

Institute Inc., 1989), to o rganize the pre dictio n

in-te rval fo r the thre e grade s o f m alignancy. Stude nt’s

t distrib utio n te st and Tuke y’s m ultiple co m pariso n

te st we re use d

to e valuate the diffe re nce s b e twe e n

the o b taine d data.

RESULTS

Clinical data

The clinical data o f the 11 patie nts are sho wn

in Tab le 1. The m e dian age was 45.7 ye ars (range

36-75). Nine patie nts we re white , 1 b lack and 1

un-kno wn. The m ale to fe m ale pro po rtio n was appro

xi-m ate ly 1:1 (6 xi-m ale and 5 fe xi-m ale ). The prixi-m ary

tu-m o r site s we re : paro tid gland (64%), hard palate ,

b ase o f to ngue , sub m axillary gland and m o uth flo o r

(9% e ach). The me an duratio n o f co mplaints was 5.6

ye ars and the m e an tum o r size was 5.3 cm . Se ve n

patie nts we re asym pto m atic and o nly 4 pre se nte d

sym pto m s at the tim e o f diagno sis.

Histopathological and immunohistochemical

find-ings

The re sults o f histo p atho lo gic al e valuatio n

sho wed 4 cases (36%) classified as lo w grade, 4 cases

Figure 4 -

Immunohistochemical studies of MEC of a salivary gland,

with a moderate number of cells positive for PCNA antigen expression.

The tumor cells were considered positive when they showed a

brownish stain. (250x magnification).

(36%) inte rm e diate and 3 case s (27%) as high m

alignancy grade . Tab le 2 pre se nts the re lative and ab

-s o lute value -s o f PCNA- p o -s itive c e ll-s in the two

analyse s pe rfo rm e d.

Whe n the m e an value s o f po sitive e xpre ssio n

o f PCNA in the diffe re nt gro ups o f histo lo gical

clas-sificatio n we re analyze d, a te nde ncy to incre ase this

e xpre ssio n was o b se rve d in case s with a high m

a-lignancy grade , as sho wn in Tab le 3. The variance

analysis using Tuke y’s te st sho we d significant

dif-fe re nce s b e twe e n the m e an value o f the high-grade

m alignancy gro up whe n co m pare d with the lo w and

inte rm e d iate grad e gro up s (P< 0.01). The se latte r

gro ups did no t pre se nt diffe re nce s b e twe e n e ach

o the r.

DISCUSSION

Altho ugh MEC was de fine d as a spe cific e

n-tity five de cade s ago , co ntro ve rsie s pe rsist co nce

rn-ing the histo patho lo gical m alignancy grade s,

sur-vival and pro gno sis in the se tum o rs.

18,19

A co m pariso n o f the re sults o f this study with

data in the lite rature sho we d agre e m e nt re garding

the mo st fre q ue nt site (paro tid gland in 64%),

2,20

_age

(fifth de cade )

20

_{and a large r pre vale nce in white}

in-dividuals.

2, 21, 22

_{The tim e take n fo r the d ise ase to}

e vo lve (5.3 ye ars) was sim ilar to that o b se rve d b y

Auclair & Ellis

2

_{and Lo yo la e t al (6.2 ye ars). In spite}

o f disagre e m e nt with so m e re po rts in the lite rature

indicating fe m ale pre do m inance ,

22-24

_{the sim ilarity}

in pro po rtio ns b etween the sexes that we fo und co

nco rds with o the r de scriptio ns in which e q ual pro

-po rtio ns we re e nco unte re d.

4,20, 25

It is wide ly acce pte d that the histo lo gical m

a-lignancy grade o f MEC is re late d to a m o re unfavo

r-ab le o utco m e in high grade tum o rs whe n co m pare d

with o the r grade s.

2, 6,19,26

_{The re is, ho we ve r, a large}

variatio n in patho lo gical crite ria use d in classifying

these tumo rs, while the evaluatio n o f the tumo r pro

-po rtio n o f cystic are as fo rms part o f all curre nt

grading syste m s. In m any case s the re is difficulty in pre

-cisely determining the cystic co mpo nent. Additio nal

classificatio n crite ria m ay b e use ful unde r the se

cir-cum stance s.

Data pre se nte d he re sho w that the e valuatio n

o f ce ll pro life ratio n using PCNA has a clo se re

la-tio nship to m alignancy grade s de te rm ine d b y

his-to patho lo gical m e tho ds.

Table 1 Clinical data on 11 case s of salivary gland mucoe pide rmoid carcinoma

-He ad and Ne ck Surge ry Se rvice - -He liópolis Hospital, São Paulo - 1978 to 1997

Ca se N º

Age (yea r)

Gender

Ra ce

Symptoms

Loca tion

Tumor Size (cm) Duration of Complaints

1 2 6 F W hite Asympto matic Paro tid 3 .0 1 4 y

2 7 5 F W hite Sympto matic Paro tid 7 .0 5 y

3 7 3 M W hite Asympto matic Submandibular 5 .0 1 y

4 4 4 M W hite Asympto matic Paro tid 1 0 .0 1 y

5 4 6 F W hite Asympto matic Base o f to ng ue 5 .0 na*

6 5 1 M Black Sympto matic Flo o r o f mo uth 2 .5 1 4 y

7 3 6 F W hite Asympto matic Paro tid 6 .0 5 y

8 4 3 M W hite Sympto matic Hard palate 2 .5 1 5 y

9 na* M na* na* Paro tid 6 .0 3 y

1 0 5 2 F W hite Sympto matic Paro tid 3 .0 1 y

1 1 5 7 M W hite Asympto matic Paro tid 1 2 .0 3 y

* na = no t available.

Table 2- Comparison be twe e n the histopathological classification and

re lative and absolute value s of p ro life ra ting c e ll nuc le a r a ntig e n (PCNA+) ce lls for all case s

1 st Eva lua tion

2 nd Eva lua tion

Histopa thologica l

PCN A+ cells

PCN A+ cells

Ca se N º

Cla ssifica tion

Tota l cells

Absolute

Rela tive

Tota l cells

Absolute

Rela tive

1 Lo w g rade 1 2 6 8 1 9 6 1 5 .5 % 1 1 8 6 1 7 2 1 4 .5 %

3 Lo w g rade 9 0 7 1 4 1 1 5 .5 % 1 1 0 9 1 5 1 1 3 .6 %

8 Lo w g rade 1 0 6 9 2 1 3 1 9 .9 % 1 1 1 6 1 7 9 1 6 .0 %

1 0 Lo w g rade 1 1 2 0 2 1 0 1 8 .8 % 1 1 1 3 1 6 1 1 4 .5 %

2 Intermediate g rade 1 1 8 0 2 4 7 2 1 .0 % 1 1 2 2 2 5 3 2 2 .6 % 4 Intermediate g rade 1 8 4 2 3 7 5 2 0 .4 % 1 1 0 9 2 7 2 2 4 .5 % 6 Intermediate g rade 1 2 3 1 4 6 2 3 7 .5 % 1 3 2 4 4 9 2 3 7 .2 % 7 Intermediate g rade 1 5 0 8 5 3 5 3 5 .5 % 1 7 6 1 6 8 3 3 8 .8 %

5 Hig h g rade 1 2 1 7 4 3 7 3 5 .9 % 1 2 2 4 5 3 7 4 3 .9 %

9 Hig h g rade 1 1 4 6 7 2 6 6 3 .4 % 1 2 0 6 9 2 1 6 8 .1 %

1. Thackray AC, Lucas RB. Tumo rs o f the majo r salivary glands. Atlas o f Tumo r Patho lo gy, 2nd Series, Fascicle 10. Washingto n DC: Armed Fo rces Institute o f Patho lo gy; 1974.

2. Auclair PL, Ellis GL. Muco epidermo id carcino ma. In: Ellis GL, Auclair PL, Gneep DR. Surgical patho lo gy o f salivary glands. Philadelphia: Saunders; 1991:269-98.

3. Franzi SA, Carvalho MB. Carcino ma muco epidermó ide avançado das glândulas salivares. Rev Bras Cancero l 1997;43(4):273-80.

4. Lo yo la AM, Araújo VC, So usa SOM, Araújo NS. Mino r salivary glands tumo rs. A retro spective study o f 164 cases in a Brazilian po pulatio n. Oral Onco l Eur J Cancer 1995;31B:197-201.

5. Se ife rt G, So b in LH. Wo rld He alth O rg anizatio n his to lo g ic al classificatio n o f tumo rs. Histo lo gical typing o f salivary gland tumo rs. Berlin: Springer; 1991:8-9.

REFERENCES

6. Seifert G, So b in LH. The Wo rld Health Organizatio n Histo lo gical classificatio n o f salivary gland tumo rs: a co mmentary o n the seco nd editio n. Cancer 1992;70:379-85.

7. Spiro RH, Huvo s AG, Berk R, Stro ng EW. Muco epidermo id carcino ma o f salivary gland o rigin. Am J Surg 1978;136:461-8.

8. Rapo po rt A, Carvalho MB, Fava AS, et al. Diagnó stico e tratamento das neo plasias das glândulas salivares meno res: estudo de 55 caso s. Rev Co l Bras Cirur 1988;5:289-93.

9. O gawa I, Miyac hi M, Takata T, Vuhahula E, Ijuhin N, Nikai H. Pro liferative activity o f salivary gland pleo mo rphic adeno ma and myo epithelio ma as evaluated by the pro liferating cell nuclear antigen (PCNA) labeling index (LI). J Oral Patho l Med 1993;22:447-50.

10. Ts uji T, Sas aki K, Kim ura Y, Yam ad a K, Mo ri M, Shino zaki F. Measurement o f pro liferating cell nuclear antigen (PCNA) and its

Table 3 - Me an pe rce ntage and dispe rsion value s of

p ro life ra ting c e ll nuc le a r a ntig e n (PCNA+) ce lls for

all patie nts and in e ach group according to the

histopathological classification

Histopathological

Statistical

Relative values of

Classification Malignancy grade

parameters

PCN A+ cells

All g ro ups n 1 1 (1 8 .9 )

Mean 3 2 .2 %

Lo w g rade g ro up n 4 (1 .6 ) Mean 1 6 .0 % * Intermediate g rade g ro up n 4 (8 .7 )

Mean 2 9 .7 % * Hig h g rade g ro up n 3 (1 4 .8 )

Mean 5 6 .9 % * * * no t sig nificant statistical difference; * * sig nificant statistical difference (P<0 .0 1 ); Standard Deviatio n g iven in parenthesis.

Co ntrary to

the results o b tained b y Tsai & Jin,

27

the data o f the pre se nt study sho we d a statistically

s ig n ific an t d iffe re n c e b e twe e n th e m alig n an c y

grade s and the e xpre ssio n o f PCNA in tum o r ce lls,

as e valuate d b y the p e rc e ntage o f p o sitive c e lls.

Tum o rs with a high grade o f m alignancy sho we d a

gre ate r pe rce ntage o f PCNA-po sitive ce lls than the

tum o rs with inte rm e diate o r lo w grade .

r e s u m o

CON TEXTO:

As características cito ló g icas e mo rfo ló g icas do Carci-no ma Muco epidermó ide, um do s critério s mais impo rtantes na aferição do seu co mpro metimento bio ló g ico e ag ressividade é a pro liferação celular. Daí, a utilização do uso de marcado res imuno histo químico s da pro liferação celular na classificação do s tumo res e na determinação do pro g nó stico e no estudo terapêutico dessas pato lo g ias.

OBJETIVO:

Analisar 1 1 caso s de carcino ma muco epidermó ide (CME) a tra vé s do a ntíg e no nuc le a r de pro life ra ç ã o c e lula r (PC N A) e determinar sua relação co m o g rau de malig nidade destes tumo res.

TIPO DE ESTUDO:

Estudo de co rrelação .

LOCAL:

Serviço de Cirurg ia de Cabeça e Pesco ço do Ho spital Helió po lis, São Paulo , Braz il.

AM OSTRA:

As lâminas de 1 1 caso s fo ram preparadas de aco rdo c o m a ro tina té c nic a do De pa rta me nto de Pa to lo g ia O ra l da Fa c uld a d e d e O d o nto lo g ia d a Unive rsid a d e d e Sã o Pa ulo , c o rre sp o nd e nd o a o c a rc ino ma muc o e p id e rmó id e p rimá rio d e g lândulas salivares, fixadas em so lução de fo rmaldeído a 1 0 % e co radas co m hemato xilina e eo sina. Apó s o preparo , as neo plasias fo ra m c la ssific a d a s e m b a ixo , inte rme d iá rio e a lto g ra u d e malig nidade, de aco rdo co m o critério de Seifert & So bin e Auclair & Ellis. As lâminas fo ram encaminhadas a estudo imuno histo químico para avaliar a po sitividade do PCN A pela técnica de streptavidina.

VARIÁVEIS ESTUDADAS:

Co rrelação entre a expressão PCN A e o g rau histo ló g ico de malig nidade do s CME de g lândulas salivares

RESULTADOS:

Haviam 4 caso s (3 6 %) de baixo g rau, 4 caso s (3 6 %) de g rau intermediário e 3 caso s (2 7 %) de alto g rau de malig nidade. Apó s o estudo co mparativo entre o s achado s histo ló g ico s e a análise imuno histo q uímic a , dife re nç a s sig nific a tiva s fo ra m o b se rva da s (P<0 ,0 1 ) para o s 3 g raus de malig nidade – 1 6 ,0 4 %, 2 6 ,9 8 % e 5 6 ,9 8 % respectivamente – da expressão do PCN A no CME.

CO N CLUSÃO :

A expressão do PCN A aumenta co m o g rau de malig nidade do CME nas g lândulas salivares.

PA LAV RA S- CH AV E:

PC N A . C a rc ino ma muc o e p id e rmó id e . G lândulas salivares.

We de r Pe re ira Cardoso, MD. Department o f Head and Neck Surgery o f Helió po lis Ho spital, São Paulo , Brazil.

Odilon Victor Porto De nardin, MD, PhD. Department o f Endo crino lo gy o f Helió po lis Ho spital, São Paulo , Brazil.

Abrão Rapoport, MD, PhD. Department o f Head and Neck Surgery o f Helió po lis Ho spital, São Paulo , Brazil.

Ve ra Cavalcanti Araújo, DDS, PhD. Patho lo gy Department o f the Dentistry Faculty, São Paulo University, São Paulo , Brazil.

Marcos Brasilino Carvalho, MD, PhD. Department o f Head and Neck Surgery o f Helió po lis Ho spital, São Paulo , Brazil.

Presented at the 6th World Congress on O ral Cancer, New Delhi, India, Feb 16th 1999

Source s of funding: This investigatio n was suppo rted by CAPES, Brazil.

Conflict inte re st: No t declared

Last re ce ive d: 25 Octo ber 1999

Acce pte d: 14 December 1999

Addre ss for corre sponde nce :

Abrão Rapo po rt

Praça Amadeu Amaral, 47 - Cj. 82 São Paulo /SP – Brasil - CEP 01327-010

publishing in fo r m a t io n

c linic al ap p lic atio n in o ral c anc e rs . lnt J O ral Maxillo fac Surg

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