Proliferating cell nuclear antigen (PCNA) in common epidermal lesions An immunohistoehemical study of proliferating eeil populations
.\
commc-i e ially available- a n t i b o d y
antigen
w a s used
to prohfc-rating
ccJI
W. A. Geary & P. H. Cooper
nttclc-ar
t o c h a r a c teri/.c anel e o n i p a r e - |)roliferatiiig
cell
p o p i i l a l i o n s ill p a r a l l i n s c - c t i o n s o f b e n i g n , ] j r c - n i a l i g i i a n t , a n d malignani
lesions ol h u m a n
toc h e n i i c a l
tec h n i c | i i e s .
c-pidc-rmis tising r o u t i n e
immtmohis-
Departments of "Pathology. ^Dermatology. University of Virginia hiealth Sciences Center. Charlottesville. Virginia. USA
T h r e e - p a l l c i i i s c m e r g e e l . , ' \ n ordered | ) a t -
tc-rn w a s I c j u n d i n | ) i u r i g o n o d i i l a r i s a n d k c - r a t o a c a n t h o m a , whc-re-m m o d c - r a t c l y a n d s t r o n g l y
positive- n u d c - i we-rc
111 a c c i n l n i t t o t t s , b a s a l - s i i p r a b a s a l thickness.
in p r o g r e s s i v e J y
dy.spltishc
pallc-rn
n i o i i s ee-ll e ' a r c i n o m a .
m o r e su|)erlic-ial c'ljidc-rmal
w a s loimd
sticingly' positive- luicle-i.
PdNA,
w i t h a |)re-poiideraiic'c- ol'
T h e s e we're- l o e a h z e - e l t o b a s a l - s t i p r a b a -
s a l /.e)nc-s t h a t w e r e - o l l e n c - x p a n d e d . the- stirfaee- w a s o f t e n
a dijjuse and
dv^jildslic
cells.
i n ac t i m e k c - r a t o s i s a n d s c | i i a -
\ u c l c - i o f c-ssc-iitially a l l d y s | ) l a s t i c e e J l s o f
b o t h e a t e - g o r i e s e-xpre-sse-el
toward
chstributc-d
ttnilorm
Tlu'ie- w a s graele-d l o s s a n d u l t i m a t e - e-xtinctieni ol
l'(!N,\ stainmg . \ IxiMil
l a y e r of r c - l a t i v c - l y
L o s s o l P ( 11\,'\
abrupt.
rc-activity
B o w c n ' s dise-asc-
c-xhibited
p a l t c - r n , wJic-rc-m l a r g e - n i u n b c - r s of i u o c l c - r a t c - l y
s l r o n g l y p o s i t i v e - iiiic Ic-i, i n r a n d o m
c-ssc-ii(ially f u l l t h i c k n c - s s c l i s t i i b n t i o n . la\c-r ol c y t o l o g i c a l l v
bland
a r r a y , wc-rc- prc-sc-nt m
In m a n y
b a s a l cc-lls, w i l h
l i e - I d s , hov\'c-VH-r, a
l a i n l cu' n o t i i i c l c - a r
s t a m i u c g , w a s l n t e - r p o s e - d bc-lwc-(-n d y s p l a s t i c ( - p i t h c - l u m i a n d clc-riiiis.
T h i s s l u e l y h a s d c - m o n s l i .ilc-el i h a l
p r o h f c - r a t i n g ec-ll p o p -
u l . i t i o n s i n ( - | ) i e l e - r m a l I c - s i o n s c a n be- assc-ssc-cl w i t h s i m p l e - , i n e-xpc-nsive- m e - t h o d s . th(-
T h e r e - we-re- e - o n s i s t e - n t dillc-rc-iic-c-s bc-twc-c-n
p r o h l c - r a t i i i g cc-ll p o p u l a t i o n s of ihc- v a r i o t i s c-nlilic-s s l u d i e - c l ,
dillc-i cMuc-s t h a t
c a n be- r e - a s o i i a b l y c o r r e - l a t e - d w i t h o t h c - r
known
c J i n i c a l , n i i c r o s c c i p i e \ a n d b i o l o g i e ' fe-ature-s o f t h e - l e s i o n s . lc-e-hiiic|iic- s h o i i l e l
provide- a n int(-re-s(ing
dive-rsc- c-tttaneoiis
(ic-aiv
This
n e - w ave-niie- f o rsttiely of
dtsc-asc-s.
\V.\, Cloopc-r l ' H . Frolifc-rating ec-ll n u c l e a r ( l ' C N A ) in
c o m m o n c-pidermal lesions. An immtmohistocJie-mical s t u d y e)f prolifc-rating ce'll |5optilations. J C u t a n ©
Munksgaard
l'athol
1992: 1 9 : 4 5 8 - 4 6 8 .
Accepted July 1. 1992
1992.
Sc-vc-ral tc-c-hiiic|iic-s a l l o w i d c - n t i f l c a t i o n a n d e m t m c - r -
nne-lear a n t i g e n
alioii
pccted,
of |)rolilc-rafing
e-ell
populatiotis
in
biohjgic
| ) i c - ] ) a r a t i o u s a n d h c u n a n t i s s u e s ( 1—3). A m o n g t h e s e are
c-ottnling
tritiatc-d
of mitolic-
thyniichiic-
Philip H. Cooper, M. D. Department of Pathology. Box 214. Universify of Virginia Health Sciences Center. Charlottensville. VA 22908, USA.
figttrcs
(4), labeling
(.')), b r o m e j d e e ) x y u r i d i i u -
with incor-
(KINA)
a|jplication
( 1 2 - 2 2 ) . As m i g h t be- c-\-
of these
tce-hniqtie-s to
httman
m a t e r i a l h a s fe)eused on t h e i r |)e)te-ntial for a s s e s s i n g the
prognosis
of m a l i g n a n t
nc-oplasms
(3).
Little
a t t e n t i o n h a s b e e n given to tlic-ir use- in s t u d y i n g t h e
p o r . i i i o n (()), nov\ ( - y t o n i e f r i c a n a l y s i s of the- S - p h a s e
te)pe)gi-apliy of p r o l i f e r a t i n g cc-ll | ) o | ) u l a l i o n s in v a r i -
lrae-tie)ii
( 7 ) , siUc-r
stains
ous disease states.
regioti.s
(8,9),
imniunohisfocliemie-al
and
of n u c l e o l a r
s l i a t i e i n e)f prcilifc-ratie)ii-asse)ciated
organi/.iiig clemon-
prote-ins s u e h as
In
this stttdy, we c-mploy a c-onmicrcially
avail-
able- nie)ne)cle)nal antiboely t h a t rc-c:c)gni/.es P C N A in
K i - ( i 7 ( I f ) , I I ) , |)lf).") ( 1 2 , I . S j , p H . ' ) ( 1 4 ) , f l u - c - y c l i n
lc)rmalin-nxe-d,
gene family preiduets (I.'), Uj), a n d p r o l i f e r a t i n g cell
(2S) tej ule-ntily, c h a r a c t e r i / . e , a n d cejmpare vhe n a -
458
|)araniii-cnibecldcd
tissue
sections
PCNA in epidermal lesions /• t^. I. ( loni 1 o l (-pidc-rmis. ( X t i O ) . \iilf: a l l p h o t o m i c r o g i a p h s arc- ol sc-cMions s t a m c - d loi P( IN.A by
iiiiniiino|>(-id\id,isc-
1(-( h n i c j lie- w It l i o n I c ciniitc-istaiii.
of p r o h l e - r a t i n g
ee-ll
p o p i i l a l i o n s in se-lc-ctcd b e i i i g u , p r c - m a h g n a n t ,
anel
and
malignant
spatial
elistribiilion
sc|tiamous
Ic-sions of h u m a n
c-pidermis.
O n l y s p a r s e - d a t a ol t h i s type- are- eiirre-ntly a v a i l a b l e iii the- lile-raliire- ( 2 1 - 2 ( ) ) .
Material and methods Case selection
s i n i | 5 l e x c h r o n i c t t s ty|)c- a b n o r n i a l i t i c - s a t thc-ii- n i a r g i u s . K . \ i i i c - | i i d c - c l b o t h p r o l i f c - i . i t i v c- a n d p a r t i a l l y i u v - o l u t e d l e s i o n s . A K v\c-rc- o f t h e - c - o n v e - n t i o n a l Iv p c - , c - x h i b i t e d well-ele-v-e-le)pc-d e y t o l c i g i e - a t y p i a iti the- b a s i l a r / o n e - , a n d c - x c - h i d e - e l l e - s t o n s v\ i t h f o c i o f s t i p e - r l l c i a l s e e o r the)se- v \ i t h I t t l l - t h i c k t u - s s d v s p l a s i a (bc)v\e-ue)iel fc-aturc-s). all Gradeu s i n g a 1 p a r t syste-111, a n d c a m e - IVeiiii s i m - d a m a g e - e l s k i n , ( i r e n v t l i p a t t e - r i i s v\e-re v a r i a b l e - a n d
inchulc-d
L p i d e - i i n a l le-sions e h o s e - u f o r s t u e l y inehiele-el p r u r i g e ) n o d i i l a r i s ( P . N , 11= 11); k e - r a t o a e a n t h o i n a ( K . \ , 11 = ! ) ) ; a c t i n i c k e - r a t o s i s ( , A K , 1 1 = 1,')); s c ( u a n i o u s cc-ll c a r c i u o m a ( S C C ' , 11= 1 I ) : a n d B o w e n ' s disc-asc- ( H I ) ,
s i M c t i o u s l v ])lc-omor|:)liic-, a t v p i c a l cc-lls a n d eithc-r n o
1 1 = 1 2 ) . ( M I S C S v \ i t l i iiiic-c|uiv-c)c-al m i c r o s c o p i c -
disc-c-rnablc- o r o n l y i n i n i m a l .tciinic- c-Iasiosis. ( i o n -
lind-
i n g s v\-e-re- s e - l e c t c - d f i - o m t h e - lile-s o f the- l ) e - p a r t m e - i i t o f P a l h o l o g y , U n i v e r s i t y e)f X ' i r g i n i a H e - a l t l i Se-ie-iie-e-s ( i c - n t c - r , f r o m m a t e - r i a l i h a l h a e l bc-e-ii p r o e e - s s e - e l a f t e - r j a i i t t a r v 1, 1 9 8 9 . L e s i o n s o f P N g c - n e - r a l l y h a d l i c h c - i i
c r a t c - r i l h r m , k e r a t i n i z i n g t t i m o r s a n d more- c-omplc-x i n l i l t r a t i v c - Ic-sions w i t h less o b v i o u s
kc-tatini/.uion.
B D c-onsiste-d ol l u l l - t h i e - k n c s s d y s p l a s i a s v \ i t h e o i i -
t r o l C])ide-rmis (11= It)) w a s obtainc-el f r o m
stin-pro-
tectcd, grosslv n o r m a l skin that acconipanic-d stirgical spc-c-inic-ns rc-sc-cted l o r n c i n d c - r n i a t o l o g i c - d i s c - a s c - .
.5 .
•
•
.•••
*.
T
ri^':.- . • '•'i--..'
nodiii.iris. (XdO).
459
Geary & Cooper S ,1
/'/i.'. .'>'. K c - t a t o a c a i i t h o n i a . ••-V
A-,
'X ^
Immunohistochemistry Mouse- nionoe;lonal antibody (PCKI; Ig(;2a) specific for P ( ; N A was jnirchased from Ne)vocasfra Labe)rate)rie-s (Cireat Britain). Visuali/atiein eif ])rimary aiitibeuly-antigen binding was ac-hie'vc-d with commercially available avidin-biotin based methe)ds (l*"Jite-/Ve-ctastaiii; Ve-etor Laboratories, Inc, Burlingame- Clalile)rnia) and diaminobenzidine- (DAB) chre)me)gen. 'I he see'ondary antibe)dy was goat antime)usc- reagent speeilic for Ig(j. Staining parame-tc-rs were- chosen after studies of the eflects of varying time-, tempc-rature, and |)rimary antibody diltttion during primary incMibatiein. (])ptimal e-onditions were- establishc-d as 1:400 primary antibeiely elilution (from a 1.0 ml stock sohition)* applied by overnight inctibation (12-14 hotirs) at 4—8°C. Secondary antibody, avidin-biotin, and DAB rc-actions we-re- performed at room tempc-rattirc-. I'ositive controls incltided sections of metastatic me-lanoma and "sattsagc-" blocks of malignant and benign brc-ast fumors. Nut cotintc-rstaining with 0.5% basic fuschin ( I s ) , 'l'his counterstain permitted liiste)le)gic ceintrol with e)nly mine)r e)l)se-uration of nuclc-ar DAB deposition. Neverthelc-ss, nnstainc-el sections were- sujie-rior lor c-stimatitig the number, distribution, and staining intc-nsity e)f j^ositive- nttcle-i. Niic-lc-ar rc-ac-tivity was assc-ssed as strong, moderate, or weak using basc-linc-s c-stablishc-d dtiring examination of sinuilfanc-oiisly sfaiiied examples of all sttidy groups. Staining was often coarsely granular in the- strong and me)dc-ratc- catc-gories, whereas wc-akly stainc-el nuclei oftc-n contained both finely granular and dilltise nttdeoplasmic staining. Nuc-leoli wc-ic- uniformly negative for PCNA. The ]5re)|5ortie)n e)f positive nuclei and their range of intensities within i5re)liferative ze)nes of each specimen were (-stimated during joint study of the material. Motphometric methocls were beyond the sce)pe and pur[3ose of the present work. Photomicrogra|)hs we-re- of uncounterstained sections that had been slaine-d, as a group, for PCNA. All ne-gativcs were processed and prints obtained unde-r identical conditie)ns. Differences in intensity e)f nuclear staining in the photomicrographs are representative of dilTercnces observed micre)scopie-ally with the- c-xce|)tieMi that
PCNA in epidermal lesions /•'if;. / . K c - r a t o a c - a i i t h o n i a . Sc-gmc-nls o f b a s a l lack iinclc-ar
l a \ c-r
staining.
-'..
'
•
• - '
..
C)\-c-ti\itig biitttc-ss is w e l l rc-pic-sc-tite-d.
(XbO).
Vi,: •'./ ,-,-.^;V' •\-
•,-j
4.
' .•'-•
i;^^/•.^-\--•
weakly stained nuclei ge-ue-rally appeared darke-r in the final |irints.
at least !)t)% of the cell population (Figs. 3, 4). A relatively homogeneous, positively stained, basilar rim, from 1 to 2 to as many as 3 cells in thickness, was thus creatc-d that consistcntlv' ontline-d the e|)iResults thelial islands and nests that comprise R,'\. IhireacCnnlrol epidermis — Nticle-i we-aklv' or moderately posi- live nuclc-i wc-rc- rare- in the basal layer. \ ge-ne-rally tive for PCNA were located haphazardly in the tinilbrm and e)b\-ie>us t-e^duction and ultimate leiss eif basal layc-r where they coni|)osc-cl lix)m a|3|:)roxinuclear staining was ce)iisistently found among cells mately .') to as many as 30"/) of the basal cell popresiding in progressively more suprabasal locations. ulation (Fig. I). Strotigiy positive tiuclei were un.•\ jiattcrn of PCNA activity similar to that just ee)mme)n. Similar |ie)sitiv-e nucle-i we-re also present dc-scribed was also pie-sent in e-])ide-imis e-e)\-ering the in the- near sn])rabasal layer. Nuele-i in the mid- and buttress at either side e)f K;\ (Fig. 4) and at excision superlieial e-|iiele-rmis were either weakly positive or, margins of six lesions. In three cases, howener, there more oftc-n, nc-gativc- for PC'NA. was abrti|M diminution of I'CN.X positivity in adPruri-iodorcagc-nl l o t dc-tc-ction o l I ) X . \ ic-pliJ l i i : 1 7 1. .S.A. I ) N . \ H o w c \ l o n u - l i \ i n s o l i d
I l l i n o i s : pr.ic-lical a s p c - c t s a n d c l i n i c . i l . i p j i l i c a t i o t i s . .Sc-niin D i a g n l'athol 19,'i'): ti: .").i. H. I rc-rc- f ) . P c - s s i o n . A . l ) c - r r i i / i i i i . \ I . I h c - s i l \ c - i - s t , i i u c - c l p r o t(-ins o l i n t c - r p h a s i c luic-ic-olar o r g a m / i - r r c - g i o n s . i sa p . i r a n i c-tc-r o f c - c - l l c h i p l i c a l i o n r a t e - . I ' . x p C c - l l R c - s 1!I(S9: I ! ! 1: I ' M . 9 . I ) c - i r u / i u i M . P c - s s i o t i . A . I rc-rc'- I ) . Q u a m i l y o l l u i c I c - o l . i i s i l v c - r - s t a t n c - c l p r o U - i n s i s rc-latc-cl l o picilifc-i . i l i n g . i c - t i \ i t \ i t i c-anc-c-r c-c-lls. L . i b l i i \ c - s l I ' I 9 O : l ) : i : I : i 7 . 10. C n c l c - s ] . l.c-mkc- 1 1 .B a i s c h I I . W . i c kc-r 11-11. S c h w a b l ^ . .Stc-m I luimau fN.i-()7. 1 1. BreuMi
I . (!c-|| c \ c i c - a n a U s i s o l a cc-ll | i r c ) l i t c - r . i t i o n - . i s s o c - i . i t c - d niiclc-.n a n l i g c - u clc-liiK-d b \ t h e - m o n e i c l e i i i . t l a i i l i b o c h I I n i i m m o l 1 9 i !I : I:!:): 1 7 1 0 . D C . ( i a l t c - i K C . M e i u e u - l e m a l . i n i i b e ) e l v K i - ( ) 7 : i t s tisc-
i n h i s t e i | ) . i l h o l e ) g \ . 1l i s t e i p a t l i e i l e i g x 1 9 9 0 : 1 7 : If!!). \'2. C l e - \ e - n g c - r C \ ' . l ' . p s t c - i n . \ l . . B a u e - i K O . M e i d t i l . u i o n e i f tliem i c - | c - , i r a n t i g e - n p I 0 : 1 a s . i l n n c M i e ) n o f e e - l l - c \ c'lc- p r o g r e - s s i e ) n . I C e - l l P I n s i e . l 1 9 ( ' ! 7 : K i l l : :VM>. l:i.
I'urite-r R R . l . a x l i c l d I . J . B i s h o p P t : . f ' . p s l e i n A l . . P . n ke-r J W . I ' i e i w e y t e ) i n e - | I IC m e - . i s n r e - m e - n t s eil p r e ) h r e - r a t i e ) n . i s s e i e i a t c d m i c l c - a r .lutigc-ii pit)."! . u \ d O X . A ccmtc-tit i n t i o n - l l o d g k i u ' s K n i p h o m a s . . \ r c l i P . i i h o l l . . i b me-el l!)('!9: I L i : 9 0 7 . I !. B e ) l t o n W l ' . . M i k i i l k a W R , H e - . i l v C ( , . S c h i n i t t l i n g R ) . K c i n o n . \ S . f . \ p r e - s s i e > n eil p t e i l i l e - r a t i e ) n a s s o e - i a t c - e l . m t i g e - n s i n t h r ccMl c y c l e - o l s \ l u h t d n i / c - e l m a m m . i h a u c c - l l s . ( \ t e n i i c - t r \ 1992: i:i: 117. 1."). W a l k e - r n i l . M a l K - r J l . . R e . l e - l o r c A c l m .\ i n t h e - d c - p e - i u l e - i u eol n i i t o s t s o t i c-otiiplc-tieiu o l ' D X . X rc-plic-.itioii. N a t u r e - 1 9 9 1 : :V>\: : i l 1. Ki. B a n d a t - a I . R , . A d a n u v . c - w s k i J P, H u n t I'. L a l ' l i a n g u c - X B . C y c l i i i .\ a n d t h e l - c - t t n o b l a s t o m a gc-tic- p r o d u c t c - e t t i i p l e - \ \ \ i t h a c e i n i m e i t i u a n s e r i p t i o n l a c - t o r . X . i l n r e - 1 9 9 1 : !i.'')2: 2 1 9 . 1 7 . M i \ a c h i K . l - ' r i t / l c t . \ l | . ' f ' a i i l ' . . \ l . . A i i t e i a t i l i b e i d v t o .i n u c - | c - a r .uitigc-ii i n p r o l i l ' c r a t i n g ec-lls. | l m n u m o l 197!i: 1 2 1 : 2 2 2 8 . I ' i . l i i a \ d R , C c ' l i s J K . .A s c - a i e h feit e l i H e - r c - i i t i a l p e ) l y p e - p t i d c s y i i t h c - s i s t h r o u g h o u t t h e - ee-ll e-\ c l c - o i l l c - l . . i c c - l l s . | C a - l l H i o l 1 9 8 0 : ! ! 1 : 79."!. 19.
B i a \ d R . l-'e-\ . S | , B c l l a t i i i j . l , . u s e - n I ' M . A r c - \ a l e . |. ( i c l i s | K . l d c - n l i r u a t i o n o l ,i n u c l c - a r a n d o l ' a c-\ t o p l a s t n i c - p o K p c - p l i d e w hose- re-lati\c- p r o p e ) r l i o n s .irc- s e ' n s i t i \ e - t o c h a u g c - s i n t h e r a t c - o l c-c-U p r o l i l c - r a t i o n . l ' ' , \ p C a - U R c - s 1 9 8 1 : \ .My. 3 1 1 .
20.
l a k a s a k i ^', l ) c - n g | - S . f a n M . M . .A i i i i e - | c - a r a n t i g c - n a s s o e i a t c - d w i t h cc-ll p r o l i l c - r a l i c i n a n d b l a s t t r a n s l o r m a t i o n . I t s d i s t t - i b i i t i o n i n s \ i i c h i d i i i z c - d c c - l l s . | l ^ . x p M e - d I!)
.\
commc-i e ially available- a n t i b o d y
antigen
w a s used
to prohfc-rating
ccJI
W. A. Geary & P. H. Cooper
nttclc-ar
t o c h a r a c teri/.c anel e o n i p a r e - |)roliferatiiig
cell
p o p i i l a l i o n s ill p a r a l l i n s c - c t i o n s o f b e n i g n , ] j r c - n i a l i g i i a n t , a n d malignani
lesions ol h u m a n
toc h e n i i c a l
tec h n i c | i i e s .
c-pidc-rmis tising r o u t i n e
immtmohis-
Departments of "Pathology. ^Dermatology. University of Virginia hiealth Sciences Center. Charlottesville. Virginia. USA
T h r e e - p a l l c i i i s c m e r g e e l . , ' \ n ordered | ) a t -
tc-rn w a s I c j u n d i n | ) i u r i g o n o d i i l a r i s a n d k c - r a t o a c a n t h o m a , whc-re-m m o d c - r a t c l y a n d s t r o n g l y
positive- n u d c - i we-rc
111 a c c i n l n i t t o t t s , b a s a l - s i i p r a b a s a l thickness.
in p r o g r e s s i v e J y
dy.spltishc
pallc-rn
n i o i i s ee-ll e ' a r c i n o m a .
m o r e su|)erlic-ial c'ljidc-rmal
w a s loimd
sticingly' positive- luicle-i.
PdNA,
w i t h a |)re-poiideraiic'c- ol'
T h e s e we're- l o e a h z e - e l t o b a s a l - s t i p r a b a -
s a l /.e)nc-s t h a t w e r e - o l l e n c - x p a n d e d . the- stirfaee- w a s o f t e n
a dijjuse and
dv^jildslic
cells.
i n ac t i m e k c - r a t o s i s a n d s c | i i a -
\ u c l c - i o f c-ssc-iitially a l l d y s | ) l a s t i c e e J l s o f
b o t h e a t e - g o r i e s e-xpre-sse-el
toward
chstributc-d
ttnilorm
Tlu'ie- w a s graele-d l o s s a n d u l t i m a t e - e-xtinctieni ol
l'(!N,\ stainmg . \ IxiMil
l a y e r of r c - l a t i v c - l y
L o s s o l P ( 11\,'\
abrupt.
rc-activity
B o w c n ' s dise-asc-
c-xhibited
p a l t c - r n , wJic-rc-m l a r g e - n i u n b c - r s of i u o c l c - r a t c - l y
s l r o n g l y p o s i t i v e - iiiic Ic-i, i n r a n d o m
c-ssc-ii(ially f u l l t h i c k n c - s s c l i s t i i b n t i o n . la\c-r ol c y t o l o g i c a l l v
bland
a r r a y , wc-rc- prc-sc-nt m
In m a n y
b a s a l cc-lls, w i l h
l i e - I d s , hov\'c-VH-r, a
l a i n l cu' n o t i i i c l c - a r
s t a m i u c g , w a s l n t e - r p o s e - d bc-lwc-(-n d y s p l a s t i c ( - p i t h c - l u m i a n d clc-riiiis.
T h i s s l u e l y h a s d c - m o n s l i .ilc-el i h a l
p r o h f c - r a t i n g ec-ll p o p -
u l . i t i o n s i n ( - | ) i e l e - r m a l I c - s i o n s c a n be- assc-ssc-cl w i t h s i m p l e - , i n e-xpc-nsive- m e - t h o d s . th(-
T h e r e - we-re- e - o n s i s t e - n t dillc-rc-iic-c-s bc-twc-c-n
p r o h l c - r a t i i i g cc-ll p o p u l a t i o n s of ihc- v a r i o t i s c-nlilic-s s l u d i e - c l ,
dillc-i cMuc-s t h a t
c a n be- r e - a s o i i a b l y c o r r e - l a t e - d w i t h o t h c - r
known
c J i n i c a l , n i i c r o s c c i p i e \ a n d b i o l o g i e ' fe-ature-s o f t h e - l e s i o n s . lc-e-hiiic|iic- s h o i i l e l
provide- a n int(-re-s(ing
dive-rsc- c-tttaneoiis
(ic-aiv
This
n e - w ave-niie- f o rsttiely of
dtsc-asc-s.
\V.\, Cloopc-r l ' H . Frolifc-rating ec-ll n u c l e a r ( l ' C N A ) in
c o m m o n c-pidermal lesions. An immtmohistocJie-mical s t u d y e)f prolifc-rating ce'll |5optilations. J C u t a n ©
Munksgaard
l'athol
1992: 1 9 : 4 5 8 - 4 6 8 .
Accepted July 1. 1992
1992.
Sc-vc-ral tc-c-hiiic|iic-s a l l o w i d c - n t i f l c a t i o n a n d e m t m c - r -
nne-lear a n t i g e n
alioii
pccted,
of |)rolilc-rafing
e-ell
populatiotis
in
biohjgic
| ) i c - ] ) a r a t i o u s a n d h c u n a n t i s s u e s ( 1—3). A m o n g t h e s e are
c-ottnling
tritiatc-d
of mitolic-
thyniichiic-
Philip H. Cooper, M. D. Department of Pathology. Box 214. Universify of Virginia Health Sciences Center. Charlottensville. VA 22908, USA.
figttrcs
(4), labeling
(.')), b r o m e j d e e ) x y u r i d i i u -
with incor-
(KINA)
a|jplication
( 1 2 - 2 2 ) . As m i g h t be- c-\-
of these
tce-hniqtie-s to
httman
m a t e r i a l h a s fe)eused on t h e i r |)e)te-ntial for a s s e s s i n g the
prognosis
of m a l i g n a n t
nc-oplasms
(3).
Little
a t t e n t i o n h a s b e e n given to tlic-ir use- in s t u d y i n g t h e
p o r . i i i o n (()), nov\ ( - y t o n i e f r i c a n a l y s i s of the- S - p h a s e
te)pe)gi-apliy of p r o l i f e r a t i n g cc-ll | ) o | ) u l a l i o n s in v a r i -
lrae-tie)ii
( 7 ) , siUc-r
stains
ous disease states.
regioti.s
(8,9),
imniunohisfocliemie-al
and
of n u c l e o l a r
s l i a t i e i n e)f prcilifc-ratie)ii-asse)ciated
organi/.iiig clemon-
prote-ins s u e h as
In
this stttdy, we c-mploy a c-onmicrcially
avail-
able- nie)ne)cle)nal antiboely t h a t rc-c:c)gni/.es P C N A in
K i - ( i 7 ( I f ) , I I ) , |)lf).") ( 1 2 , I . S j , p H . ' ) ( 1 4 ) , f l u - c - y c l i n
lc)rmalin-nxe-d,
gene family preiduets (I.'), Uj), a n d p r o l i f e r a t i n g cell
(2S) tej ule-ntily, c h a r a c t e r i / . e , a n d cejmpare vhe n a -
458
|)araniii-cnibecldcd
tissue
sections
PCNA in epidermal lesions /• t^. I. ( loni 1 o l (-pidc-rmis. ( X t i O ) . \iilf: a l l p h o t o m i c r o g i a p h s arc- ol sc-cMions s t a m c - d loi P( IN.A by
iiiiniiino|>(-id\id,isc-
1(-( h n i c j lie- w It l i o n I c ciniitc-istaiii.
of p r o h l e - r a t i n g
ee-ll
p o p i i l a l i o n s in se-lc-ctcd b e i i i g u , p r c - m a h g n a n t ,
anel
and
malignant
spatial
elistribiilion
sc|tiamous
Ic-sions of h u m a n
c-pidermis.
O n l y s p a r s e - d a t a ol t h i s type- are- eiirre-ntly a v a i l a b l e iii the- lile-raliire- ( 2 1 - 2 ( ) ) .
Material and methods Case selection
s i n i | 5 l e x c h r o n i c t t s ty|)c- a b n o r n i a l i t i c - s a t thc-ii- n i a r g i u s . K . \ i i i c - | i i d c - c l b o t h p r o l i f c - i . i t i v c- a n d p a r t i a l l y i u v - o l u t e d l e s i o n s . A K v\c-rc- o f t h e - c - o n v e - n t i o n a l Iv p c - , c - x h i b i t e d well-ele-v-e-le)pc-d e y t o l c i g i e - a t y p i a iti the- b a s i l a r / o n e - , a n d c - x c - h i d e - e l l e - s t o n s v\ i t h f o c i o f s t i p e - r l l c i a l s e e o r the)se- v \ i t h I t t l l - t h i c k t u - s s d v s p l a s i a (bc)v\e-ue)iel fc-aturc-s). all Gradeu s i n g a 1 p a r t syste-111, a n d c a m e - IVeiiii s i m - d a m a g e - e l s k i n , ( i r e n v t l i p a t t e - r i i s v\e-re v a r i a b l e - a n d
inchulc-d
L p i d e - i i n a l le-sions e h o s e - u f o r s t u e l y inehiele-el p r u r i g e ) n o d i i l a r i s ( P . N , 11= 11); k e - r a t o a e a n t h o i n a ( K . \ , 11 = ! ) ) ; a c t i n i c k e - r a t o s i s ( , A K , 1 1 = 1,')); s c ( u a n i o u s cc-ll c a r c i u o m a ( S C C ' , 11= 1 I ) : a n d B o w e n ' s disc-asc- ( H I ) ,
s i M c t i o u s l v ])lc-omor|:)liic-, a t v p i c a l cc-lls a n d eithc-r n o
1 1 = 1 2 ) . ( M I S C S v \ i t l i iiiic-c|uiv-c)c-al m i c r o s c o p i c -
disc-c-rnablc- o r o n l y i n i n i m a l .tciinic- c-Iasiosis. ( i o n -
lind-
i n g s v\-e-re- s e - l e c t c - d f i - o m t h e - lile-s o f the- l ) e - p a r t m e - i i t o f P a l h o l o g y , U n i v e r s i t y e)f X ' i r g i n i a H e - a l t l i Se-ie-iie-e-s ( i c - n t c - r , f r o m m a t e - r i a l i h a l h a e l bc-e-ii p r o e e - s s e - e l a f t e - r j a i i t t a r v 1, 1 9 8 9 . L e s i o n s o f P N g c - n e - r a l l y h a d l i c h c - i i
c r a t c - r i l h r m , k e r a t i n i z i n g t t i m o r s a n d more- c-omplc-x i n l i l t r a t i v c - Ic-sions w i t h less o b v i o u s
kc-tatini/.uion.
B D c-onsiste-d ol l u l l - t h i e - k n c s s d y s p l a s i a s v \ i t h e o i i -
t r o l C])ide-rmis (11= It)) w a s obtainc-el f r o m
stin-pro-
tectcd, grosslv n o r m a l skin that acconipanic-d stirgical spc-c-inic-ns rc-sc-cted l o r n c i n d c - r n i a t o l o g i c - d i s c - a s c - .
.5 .
•
•
.•••
*.
T
ri^':.- . • '•'i--..'
nodiii.iris. (XdO).
459
Geary & Cooper S ,1
/'/i.'. .'>'. K c - t a t o a c a i i t h o n i a . ••-V
A-,
'X ^
Immunohistochemistry Mouse- nionoe;lonal antibody (PCKI; Ig(;2a) specific for P ( ; N A was jnirchased from Ne)vocasfra Labe)rate)rie-s (Cireat Britain). Visuali/atiein eif ])rimary aiitibeuly-antigen binding was ac-hie'vc-d with commercially available avidin-biotin based methe)ds (l*"Jite-/Ve-ctastaiii; Ve-etor Laboratories, Inc, Burlingame- Clalile)rnia) and diaminobenzidine- (DAB) chre)me)gen. 'I he see'ondary antibe)dy was goat antime)usc- reagent speeilic for Ig(j. Staining parame-tc-rs were- chosen after studies of the eflects of varying time-, tempc-rature, and |)rimary antibody diltttion during primary incMibatiein. (])ptimal e-onditions were- establishc-d as 1:400 primary antibeiely elilution (from a 1.0 ml stock sohition)* applied by overnight inctibation (12-14 hotirs) at 4—8°C. Secondary antibody, avidin-biotin, and DAB rc-actions we-re- performed at room tempc-rattirc-. I'ositive controls incltided sections of metastatic me-lanoma and "sattsagc-" blocks of malignant and benign brc-ast fumors. Nut cotintc-rstaining with 0.5% basic fuschin ( I s ) , 'l'his counterstain permitted liiste)le)gic ceintrol with e)nly mine)r e)l)se-uration of nuclc-ar DAB deposition. Neverthelc-ss, nnstainc-el sections were- sujie-rior lor c-stimatitig the number, distribution, and staining intc-nsity e)f j^ositive- nttcle-i. Niic-lc-ar rc-ac-tivity was assc-ssed as strong, moderate, or weak using basc-linc-s c-stablishc-d dtiring examination of sinuilfanc-oiisly sfaiiied examples of all sttidy groups. Staining was often coarsely granular in the- strong and me)dc-ratc- catc-gories, whereas wc-akly stainc-el nuclei oftc-n contained both finely granular and dilltise nttdeoplasmic staining. Nuc-leoli wc-ic- uniformly negative for PCNA. The ]5re)|5ortie)n e)f positive nuclei and their range of intensities within i5re)liferative ze)nes of each specimen were (-stimated during joint study of the material. Motphometric methocls were beyond the sce)pe and pur[3ose of the present work. Photomicrogra|)hs we-re- of uncounterstained sections that had been slaine-d, as a group, for PCNA. All ne-gativcs were processed and prints obtained unde-r identical conditie)ns. Differences in intensity e)f nuclear staining in the photomicrographs are representative of dilTercnces observed micre)scopie-ally with the- c-xce|)tieMi that
PCNA in epidermal lesions /•'if;. / . K c - r a t o a c - a i i t h o n i a . Sc-gmc-nls o f b a s a l lack iinclc-ar
l a \ c-r
staining.
-'..
'
•
• - '
..
C)\-c-ti\itig biitttc-ss is w e l l rc-pic-sc-tite-d.
(XbO).
Vi,: •'./ ,-,-.^;V' •\-
•,-j
4.
' .•'-•
i;^^/•.^-\--•
weakly stained nuclei ge-ue-rally appeared darke-r in the final |irints.
at least !)t)% of the cell population (Figs. 3, 4). A relatively homogeneous, positively stained, basilar rim, from 1 to 2 to as many as 3 cells in thickness, was thus creatc-d that consistcntlv' ontline-d the e|)iResults thelial islands and nests that comprise R,'\. IhireacCnnlrol epidermis — Nticle-i we-aklv' or moderately posi- live nuclc-i wc-rc- rare- in the basal layer. \ ge-ne-rally tive for PCNA were located haphazardly in the tinilbrm and e)b\-ie>us t-e^duction and ultimate leiss eif basal layc-r where they coni|)osc-cl lix)m a|3|:)roxinuclear staining was ce)iisistently found among cells mately .') to as many as 30"/) of the basal cell popresiding in progressively more suprabasal locations. ulation (Fig. I). Strotigiy positive tiuclei were un.•\ jiattcrn of PCNA activity similar to that just ee)mme)n. Similar |ie)sitiv-e nucle-i we-re also present dc-scribed was also pie-sent in e-])ide-imis e-e)\-ering the in the- near sn])rabasal layer. Nuele-i in the mid- and buttress at either side e)f K;\ (Fig. 4) and at excision superlieial e-|iiele-rmis were either weakly positive or, margins of six lesions. In three cases, howener, there more oftc-n, nc-gativc- for PC'NA. was abrti|M diminution of I'CN.X positivity in adPruri-iodorcagc-nl l o t dc-tc-ction o l I ) X . \ ic-pliJ l i i : 1 7 1. .S.A. I ) N . \ H o w c \ l o n u - l i \ i n s o l i d
I l l i n o i s : pr.ic-lical a s p c - c t s a n d c l i n i c . i l . i p j i l i c a t i o t i s . .Sc-niin D i a g n l'athol 19,'i'): ti: .").i. H. I rc-rc- f ) . P c - s s i o n . A . l ) c - r r i i / i i i i . \ I . I h c - s i l \ c - i - s t , i i u c - c l p r o t(-ins o l i n t c - r p h a s i c luic-ic-olar o r g a m / i - r r c - g i o n s . i sa p . i r a n i c-tc-r o f c - c - l l c h i p l i c a l i o n r a t e - . I ' . x p C c - l l R c - s 1!I(S9: I ! ! 1: I ' M . 9 . I ) c - i r u / i u i M . P c - s s i o t i . A . I rc-rc'- I ) . Q u a m i l y o l l u i c I c - o l . i i s i l v c - r - s t a t n c - c l p r o U - i n s i s rc-latc-cl l o picilifc-i . i l i n g . i c - t i \ i t \ i t i c-anc-c-r c-c-lls. L . i b l i i \ c - s l I ' I 9 O : l ) : i : I : i 7 . 10. C n c l c - s ] . l.c-mkc- 1 1 .B a i s c h I I . W . i c kc-r 11-11. S c h w a b l ^ . .Stc-m I luimau fN.i-()7. 1 1. BreuMi
I . (!c-|| c \ c i c - a n a U s i s o l a cc-ll | i r c ) l i t c - r . i t i o n - . i s s o c - i . i t c - d niiclc-.n a n l i g c - u clc-liiK-d b \ t h e - m o n e i c l e i i i . t l a i i l i b o c h I I n i i m m o l 1 9 i !I : I:!:): 1 7 1 0 . D C . ( i a l t c - i K C . M e i u e u - l e m a l . i n i i b e ) e l v K i - ( ) 7 : i t s tisc-
i n h i s t e i | ) . i l h o l e ) g \ . 1l i s t e i p a t l i e i l e i g x 1 9 9 0 : 1 7 : If!!). \'2. C l e - \ e - n g c - r C \ ' . l ' . p s t c - i n . \ l . . B a u e - i K O . M e i d t i l . u i o n e i f tliem i c - | c - , i r a n t i g e - n p I 0 : 1 a s . i l n n c M i e ) n o f e e - l l - c \ c'lc- p r o g r e - s s i e ) n . I C e - l l P I n s i e . l 1 9 ( ' ! 7 : K i l l : :VM>. l:i.
I'urite-r R R . l . a x l i c l d I . J . B i s h o p P t : . f ' . p s l e i n A l . . P . n ke-r J W . I ' i e i w e y t e ) i n e - | I IC m e - . i s n r e - m e - n t s eil p r e ) h r e - r a t i e ) n . i s s e i e i a t c d m i c l c - a r .lutigc-ii pit)."! . u \ d O X . A ccmtc-tit i n t i o n - l l o d g k i u ' s K n i p h o m a s . . \ r c l i P . i i h o l l . . i b me-el l!)('!9: I L i : 9 0 7 . I !. B e ) l t o n W l ' . . M i k i i l k a W R , H e - . i l v C ( , . S c h i n i t t l i n g R ) . K c i n o n . \ S . f . \ p r e - s s i e > n eil p t e i l i l e - r a t i e ) n a s s o e - i a t c - e l . m t i g e - n s i n t h r ccMl c y c l e - o l s \ l u h t d n i / c - e l m a m m . i h a u c c - l l s . ( \ t e n i i c - t r \ 1992: i:i: 117. 1."). W a l k e - r n i l . M a l K - r J l . . R e . l e - l o r c A c l m .\ i n t h e - d c - p e - i u l e - i u eol n i i t o s t s o t i c-otiiplc-tieiu o l ' D X . X rc-plic-.itioii. N a t u r e - 1 9 9 1 : :V>\: : i l 1. Ki. B a n d a t - a I . R , . A d a n u v . c - w s k i J P, H u n t I'. L a l ' l i a n g u c - X B . C y c l i i i .\ a n d t h e l - c - t t n o b l a s t o m a gc-tic- p r o d u c t c - e t t i i p l e - \ \ \ i t h a c e i n i m e i t i u a n s e r i p t i o n l a c - t o r . X . i l n r e - 1 9 9 1 : !i.'')2: 2 1 9 . 1 7 . M i \ a c h i K . l - ' r i t / l c t . \ l | . ' f ' a i i l ' . . \ l . . A i i t e i a t i l i b e i d v t o .i n u c - | c - a r .uitigc-ii i n p r o l i l ' c r a t i n g ec-lls. | l m n u m o l 197!i: 1 2 1 : 2 2 2 8 . I ' i . l i i a \ d R , C c ' l i s J K . .A s c - a i e h feit e l i H e - r c - i i t i a l p e ) l y p e - p t i d c s y i i t h c - s i s t h r o u g h o u t t h e - ee-ll e-\ c l c - o i l l c - l . . i c c - l l s . | C a - l l H i o l 1 9 8 0 : ! ! 1 : 79."!. 19.
B i a \ d R . l-'e-\ . S | , B c l l a t i i i j . l , . u s e - n I ' M . A r c - \ a l e . |. ( i c l i s | K . l d c - n l i r u a t i o n o l ,i n u c l c - a r a n d o l ' a c-\ t o p l a s t n i c - p o K p c - p l i d e w hose- re-lati\c- p r o p e ) r l i o n s .irc- s e ' n s i t i \ e - t o c h a u g c - s i n t h e r a t c - o l c-c-U p r o l i l c - r a t i o n . l ' ' , \ p C a - U R c - s 1 9 8 1 : \ .My. 3 1 1 .
20.
l a k a s a k i ^', l ) c - n g | - S . f a n M . M . .A i i i i e - | c - a r a n t i g c - n a s s o e i a t c - d w i t h cc-ll p r o l i l c - r a l i c i n a n d b l a s t t r a n s l o r m a t i o n . I t s d i s t t - i b i i t i o n i n s \ i i c h i d i i i z c - d c c - l l s . | l ^ . x p M e - d I!)
