Sao Paulo Med. J. vol.113 número1

A lb in a M e ssia s d e A lm e id a M ila n i A lte m a n i, A g ricio C re sp o , Izild a A p a re cid a C a rd in a lli, D e n ise N o ra to

N ucleolar O rganizer R egion (N O R s) in pseudocarcinom atous

hyperplasia and squam ous cell carcinom a of the oral m ucosa

Department

of Pathology, Faculdade

de Medicina,

Universidade Estadual de Camp in as - Siio Paulo, Brazil

C ounts of nucleolar regions (N O R s) dem onstrated by a silver staining technique in paraffin sections, have been used to distinguish benign from m alignant lesions. A gN O R s w ere studied in 24 biopsies from oral cavity (5 cases of norm al oral m ucosa, 5 of

pseudocarcinom atous hyperplasia and 14 of squam ous cell carcinom a, subdivided according to degree of differentiation: 5 grade

1, 5 grade 2 and 4 grade 3) to find w hether they w ere helpful in distinguishing pseudocarcinom atous hyperplasia due to chronic parasitic infections from squam ous cell carcinom a. T w o m ethods of counting A gN O R s w ere used: (A ) a sim pler one w hich counts nucleolar clusters (A gN U ) and satellite A gN O R s and the other (8) counting all individual A gN O R s, including those w ithin A gN U s. In both m ethods the low est m ean values w ere observed for grade 3 carcinom a, w hile the highest belonged to grade 2 carcinom a. T he

sim pler m ethod (A ) w as the m ost useful because A gN U counts show ed significant difference w hen pseudocarcinom atous hyperplasia w as com pared w ith grade 1 and 2 carcinom as, w hich are the m ost difficult to discrim inate from it. H ow ever the

overlapping of values render the technique of lim ited use in individual cases.

U N IT E R M S : A gN O R s. O ral carcinom a. M outh. O ral m ucosa. P seudocarcinom atous hyperplasia. S quam ous cell carcinom a.

IN T R O D U C T IO N

P

re s p ira to rys e u d o c a rc in o m a to u sa n d d ig e s tiv eh y p e rp la s iatra c ts is c o m m o no f th e u p p e rin d e v e lo p in g c o u n trie s d u e to c h ro n ic p a ra s itic

in fe c tio n s , p a rtic u la rly p a ra c o c c id io d o m y c o s is a n d

le is h m a n io s is , a n d m a y s im u la te c a rc in o m a c lin ic a lly

a n d h is to lo g ic a lly (7 )

A d d re ss fo r co rre sp o n d e n ce :

A lb in a M e ssia s d e A lm e id a M ila n i A lte m a n i

D e p a rta m e n to d e A n a to m ia P a to l6 g ica , F C M -U N IC A M P C a ixa P o sta l 6 1 1 1

C a m p in a s - S P - B ra sil- C E P 1 3 0 8 1 -9 7 0

C o u n ts o f n u c le o la r o rg a n iz e r re g io n s (N O R s )

d e m o n s tra te d b y a s ilv e r s ta in in g te c h n iq u e in p a ra ffin

s e c tio n s , h a v e b e e n u s e d to d is tin g u i's h b e n ig n fro m

m a lig n a n t le s io n s (3 ,4 ,8 ,1 1 ). N O R s a re c h ro m o s o m a l

s e g m e n ts in w h ic h rib o s o m a l R N A (rR N A ) is e n c o d e d

(1 3 ). T h e y a re re s p o n s ib le fo r th e d e v e lo p m e n t o f n u c le o li,

in to w h ic h th e y p ro je c t la rg e lo o p s o f D N A (1 2 ).

T h e re is o n ly o n e p re v io u s s tu d y a p p ly in g A g N O R s

c o u n ts to d is tin g u is h p s e u d o -c a rc in o m a to u s h y p e rp la s ia

a n d o ra l s q u a m o u s c e ll c a rc in o m a (2 ). H o w e v e r th e a u th o rs

d id n o t c o n s id e r th e d e g re e o f d iffe re n tia tio n o f th e

s q u a m o u s c e ll c a rc in o m a in th e ir s tu d y . T h e w e

ll-d iffe re n tia te ll-d c a rc in o m a is th e m o s t d iffic u lt to

d is c rim in a te fro m p s e u d o c a rc in o m a to u s h y p e rp la s ia (7 ).

Q u r o b je c tiv e w a s to e x a m in e w h e th e r A g N O R s

re lia b ly d is tin g u is h th e s e tw o d is e a s e s .

MATERIAL AND METHODS

The

material

consisted

of

24

biopsies

from the oral

cavity;

5 samples

of

normal

oral mucosa,

5 cases of

pseudocarcinomatous

hyperplasia

due

to

paracoccidioidomycosis

and

14 of squamous

cell

carcinoma.

This latter was subdivided

according

to degree

of differentiation

in: grade

1 (5 cases)

- carcinomatous

cells

were

predominantly

of mature

squamous

type

showing

relatively

slight atypia,

grade

2

(5 cases) - many

carcinomatous

cells were atypical

and devoid of prickles,

grade 3 (4 cases) - all carcinomatous

cells were atypical

without

recognizable

intercellular

bridges

and

keratinization

was absent.

All specimens

were

formalin-fixed.

Paraffin-embedded

sections

3 urn-thick

were

stained

with the AgNOR

procedure,

according

Ploton

et

al (10), with incubation

time of 30 min. The sections were

viewed under oil immersion

(C 100) by a single observer

(AM A).

One hundred

nuclei

were randomly

examined,

but in normal

mucosa, pseudo-carcinomatous

hyperplasia

and grade 1 carcinoma

only basal

and parabasal cells were

taken into account.

Two methods

of counting

were used.

In the simpler method (A) nucleolar

clusters were counted

as a single

AgNOR,

irrespective

of the number

of dots

within the nucleolus,

and so this gave a total representing

the number

of dissociated,

satellite

AgNORs

(sAgNORs)

and nucleolar

clusters

(AgNUS).

The second method (B)

involved

counting

total

AgNORs,

including

those

aggregated

- within

AgNUS

clusters.

The

data

were

analyzed

by the Kruskall- W allis test and W ilcoxon

sum

rank test.

RESULTS

The findings in each of the five diagnostic

categories,

using both counting

methods

are shown

in table

1. All

groups showed a wide variation

in their counts (Fig. 1 and

2). The highest

means

were of the grade 2 carcinoma

in

both methods, while the lowest means were of the grade 3

carcinoma.

In relation

to the

method

A the

AgNUS

counts

showed significant

difference

when pseudocarcinomatous

hyperplasia

was compared

with grade

1 carcinoma,

grade

2 carcinoma

and normal

mucosa,

but not with

grade

3

carcinoma.

This significant

difference

was due to lower

mean

of

AgNU

found

in

pseudocarcinomatous

hyperplasia.

However

neither

total AgNORs

counts

nor

sAgNORs

counts

were

helpful

to

distinguish

pseudocarcinomatous

hyperplasia

from

grade

1

carcinoma.

The former

type

of

counting

showed

significant

difference

only between pseudocarcinomatous

hyperplasia

and grade

2

carcinoma,

while

the latter did

not show ,significant differences

among

any groups

at all.

Concerning

method B the means of AgNORs

counts were

not significantly

different

between

pseudocarcinomatous

hyperplasia

and the other

groups,

except

for grade

3

carcinoma

which had the lowest mean of AgNORs.

The morphological

study

of the AgNORs

showed

that only grade 2and 3 carcinomas

had distinctive

patterns.

Grade

2

carcinoma

presented

several AgNUS

of different

sizes and shapes

and usually

rich in dots, together

with

frequently

dissociated

satellite AgNORs.

In contrast grade

3 carcinoma

usually

had only one AgNU

with fe~

or no

Table 1

Mean

:t

SO AgNOR counts in normal mucosa and

o 'ra l

lesion

D ia g n o s tic g ro u p A g N O R A g N U s A g N O R A g N O R T

N o rm a l m u c o s a

10.82 :t 3.58

2.26:t 0.34

0.57 :t 0.25

2.83 :t 0.45

P s e u d o c a rc in o m a to u s h y p e rp la s ia

11.60 :t 2.34

1.73:t0.13

0.42 :t 0.18

2.15 :t 0.23

G ra d e

1

C a rc in o m a

' 9.59 :t 2.40

2.42 :t 0.99

0.46 :t 0.17

2.87 :t 1.12

G ra d e

2

C a rc in o m a

13.47 :t 2.38

2.95 :t 0.48

1.53 :t 0.71

4.48 :t 1.00

G ra d e

3

C a rc in o m a

4.99:t

1.43

1.53 :t 0.18

0.38:t0.11

1.91

:t

0.26

~

..,.

2 0

1 $

-

.-1 0

X

I

-X

!s '

- or- o

X X

X

r

X

t-

X

~

A B C D E ~_~

A 8 C D E

Figure 1 -

M e a n A g N O R c o u n ts p e r c a s e in e a c h g ro u p s tu d ie d . A - N o rm a l m u c o s a , B - p s e u d o c a rc io n o m a to u s h y p e rp la s ia , C

-g ra d e 1 c a rc in o m a , D - -g ra d e 2 c a rc in o m a , E - -g ra d e 3 c a rc in o m a .

d o ts ( A g N O R s ) in s id e it. P s e u d o c a r c in o m a to u s

h y p e r p la s ia a n d g r a d e Ic a r c in o m a d id n o t s h o w d is tin c tiv e m o r p h o lo g ic a l p a tte r n s o f A g N U s o r A g N O R s . B o th h a d

f e w n u c le o li, u s u a lly in c r e a s e d in s iz e , r ic h in d o ts a n d

r a r e d is s o c ia te d A g N O R s ( F ig . 3 ) .

DISCUSSION

P s e u d o c a r c in o m a to u s h y p e r p la s ia m a y o c c u r in s k in

a n d m u c o u s s u r f a c e s a s s o c ia te d to c h r o n ic p r o lif e r a tiv e

in f la m m a to r y p r o c e s s e s , e d g e s o f c h r o n ic u lc e r s a n d

g r a n u la r c e ll tu m o r ( 7 ) . T h e h is to lo g ic a l s tu d y o f th e

le s io n s h o w s ir r e g u la r p r o lif e r a tio n o f th e e p ith e liu m , w ith

in v a s io n o f th e s u b m u c o s a b y e p ith e lia l m a s s e s a n d

s tr a n d s , w ith h o r n - p e a r l f o r m a tio n a n d f r e q u e n tly

n u m e r o u s m ito tic f ig u r e s . M u ltip le b io p s ie s o f th e s e

le s io n s a n d d e ta ile d c lin ic a l d a ta m a y b e n e c e s s a r y f o r

d if f e r e n tia tio n o f s q u a m o u s c e ll c a r c in o m a , p a r tic u la r ly

g r a d e I ( w e ll- d if f e r e n tia te d ) c a r c in o m a , w h ic h s h o w s r e la tiv e ly s lig h t c e llu la r a ty p ia s ( 7 ) .

I n f e c tio u s d is e a s e s c a u s in g c h r o n ic p r o lif e r a tiv e

in f la m m a to r y p r o c e s s a s s o c ia te d to p s e u d o c a r c in o m a to u s

h y p e r p la s ia in m u c o u s m e m b r a n e s o f th e u p p e r d ig e s tiv e

a n d r e s p ir a to r y tr a c ts o c c u r f r e q u e n tly in d e v e lo p in g

c o u n tr ie s . T h e h is to lo g ic a l d if f e r e n tia tio n o f th is ty p e o f

Figure 2 -

M e a n A g N U a n d s A g N O R c o u n ts p e r c a s e in e a c h g ro u p s tu d ie d . A - n o rm a l m u c o s a , B - p s e u d o c a rc in o m a to u s h y p e rp la s ia , C - g ra d e 1 c a rc in o m a , D - g ra d e 2 c a rc in o m a , E -g ra d e 3 c a rc in o m a .

p s e u d o c a r c in o m a to u s h y p e r p la s ia a n d w e ll d if f e r e n tia te d

c a r c in o m a is a c o m m o n p r o b le m in r o u tin e s u r g ic a l

p a th o lo g y a n d r e lie s o n th e id e n tif ic a tio n o f in f e c tio u s

a g e n ts in s m a ll s p e c im e n s o f b io p s ie s .

C h u n g p a n ic h a n d S m ith ( 2 ) s u g g e s te d th a t th e

A g N O R c o u n ts c o u ld b e u s e d a s a r e lia b le in d e x f o r

d is tin g u is h in g o r a l s q u a m o u s c e ll c a r c in o m a a n d

p s e u d o c a r c in o m a to u s h y p e r p la s ia d u e to p a p illa r y

h y p e r p la s ia , d e n tu r e in d u c e d f ib r o u s h y p e r p la s ia a n d

g r a n u la r c e ll tu m o r . T h e a u th o r s c o u n te d a ll A g N 'O R d o ts

p e r n u c le u s a n d f o u n d a s ig n if ic a n tly lo w e r m e a n o f

A g N O R c o u n ts in p s e u d o c a r c in o m a to u s h y p e r p la s ia .

H o w e v e r w e f o u n d th a t A g N O R c o u n ts w e r e o f

lim ite d v a lu e to ,d is tin g u is h p s e ~ d o c a r c in o m a to u s

h y p e r p la s ia o f in f e c tio n s o r ig in a n d g r a d e I a n d 2 c a r C I n o m a .

I t s h o u ld b e n o te d th a t w e u s e d tw o m e th o d s o f

A g N O R s c o u n ts : o n e o f th e m is s im p le r a n d m o r e e a s ily

r e p r o d u c ib le a n d th e o th e r m o r e tim e - c o n s u m in g a n d m o r e

d if f ic u lt to r e p r o d u c e ( 6 ,9 ) , s im ila r to o n e u s e d b y

C h u n g p a n ic h a n d S m ith . T h e s im p le r m e th o d , w h ic h

c o u n ts n u c le o la r c lu s te r s ( A g N U ) a s a s in g le A g N O R w a s

th e m o s t h e lp f u l to d is c r im in a te b e tw e e n

p s e u d o c a r c in o m a to u s h y p e r p la s ia a n d g r a d e 1 a n d 2

c a r c in o m a . T h e m e a n A g N U S c o u n ts w a s s ig n if ic a n tly

lo w e r in th e f o r m e r le s io n . T h e m o r e tim e - c o n s u m in g

m e th o d , c o u n tin g to ta l A g N O R s d o ts , in c lu d in g th o s e

Figure

3 -

P s e u d o c a rc in o m a to u s h y p e rp la s ia (A ) a n d g ra d e 1 c a rc in o m a (8 ) d id n o t s h o w d is tin c tiv e m o rp h o lo g ic a l p a tte rn s o f A g N U s o r A g N O R s .

a g g re g a te d w ith in A g N U c lu s te rs d id n o t s h o w s ig n ific a n t d iffe re n c e b e tw e e n p s e u d o c a rc in o m a to u s h y p e rp la s ia a n d g ra d e 1 a n d 2 c a rc in o m a . T h e s im p le r m e th o d h a s b e e n a ls o u s e fu l in o th e r o rg a n s a n d tu m o rs (1 ,5 ,8 ).

H o w e v e r, in s p ite o f th e m e a n A g N U c o u n ts b e in g s ig n ific a n tly lo w e r in p s e u d o c a rc in o m a to u s h y p e rp la s ia , w e fo u n d s o m e o v e rla p p in g v a lu e s b e tw e e n th e g ro u p s . T h e re fo re th is in d e x w a s o f lim ite d u s e fo r th e d ia g n o s is o f in d iv id u a l c a s e s .

In a d d itio n , w e d id n o t o b s e rv e m o rp h o lo g ic a l d iffe re n c e s o f A g N O R s a n d A g N U S b e tw e e n p s e u d o c a rc in o m a to u s h y p e rp la s ia a n d g ra d e Ic a rc in o m a , w h ic h a re th e m o s t d iffic u lt to d is tin g u is h in s p e c im e n s o f s u rg ic a l p a th o lo g y .

In c o n c lu s io n , A g N O R s c o u n ts w e re o f lim ite d v a lu e fo r th e d i ffe re n ti a l d i a g n o s i s b e tw e e n p s e u d o c a rc in o m a to u s h y p e rp la s ia o f in fe c tio u s o rig in a n d s q u a m o u s c e ll c a rc in o m a o f th e o ra l c a v ity .

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",""",~~-. mw._~