Abs ac The cell kine ic o p os a ic in aepi helial
neoplasia (PIN) is poo ly unde s ood. He ein we epo
he kine ic pa e n o PIN, bo h no associa ed (p ima y)
and associa ed (seconda y) wi h coexis en in asi e ca -
cinoma (PCa). Su gical specimens collec ed in 20 cases
o p ima y PIN, 20 o seconda y PIN and 20 o PCa
we e s udied by MIB-1 immunos aining, in si u end-
labeling (ISEL) and DNA his og am analysis, and he
cell densi y in each case was es ima ed using he o mula
N=(nπ/4)2. Fi y high-powe ields (HPF), o he com-
ple e lesion i smalle , we e sc eened in each lesion, and
bo h mean and s anda d de ia ion we e eco ded. S a is-
ical di e ences we e s udied by means o Fishe ’s exac
es . ISEL indices we e signi ican ly (P<0.0001) lowe in
PCa (0.1±0.3) han in p ima y PIN (0.5±0.3), while he
MIB-I indices we e simila in bo h condi ions (P=0.56).
S a is ically signi ican di e ences we e also de ec ed
o bo h MIB-1 and ISEL indices when seconda y
PIN (MIB-1 1.9±0.7, ISEL 3.7±3.3) was compa ed
wi h p ima y PIN (MIB-1 2.5±2.1, ISEL 0.5±0.3) and
PCa (P<0.0001). In e ms o cellula i y, p ima y PIN
(26.3±7.1) e ealed sco es signi ican ly lowe (P<0.0001)
han hose eco ded in PCa (39.0±8.8) and seconda y
PIN (32.9±14.3). In conclusion, ea ly p os a ic umo is
mainly de ined by down- egula ed apop osis a he han
by inc eased p oli e a ion. Seconda y PIN displays
unique kine ic ea u es sugges ing an e ol ed s age o
p ima y PIN.
Key wo ds P os a ic in aepi helial neoplasm ·
P ecance ous lesion · In aduc al ex ension · Cell kine ics ·
DNA-ploidy
In oduc ion
Cu en ly, high-g ade p os a ic in aepi helial neoplasia
(PIN) is he mos likely p ecu so o in asi e p os a ic
ca cinoma (PCa) [2, 3] and has been epo ed bo h as an
isola ed inding (p ima y PIN) and coexis ing wi h in a-
si e adenoca cinomas (seconda y PIN) [4, 22].
The no mal cellula u no e is main ained by a s ic
balance be ween p oli e a ion and apop osis [27, 43],
s udied by se e al echniques including immunohis o-
chemis y, in si u end-labeling (ISEL) o agmen ed
DNA, and DNA-ploidy analysis. Con o e sial esul s
ha e been epo ed on cell kine ics in PIN because o he
he e ogenei y o cases (especially ega ding o al and o-
gen abla ion) [32, 34, 35] and he di e si y o echniques
and quan i ica ion me hods o bo h p oli e a ing cells
[23, 33, 45] and apop o ic cells [18, 31–34, 47]. Addi-
ionally, no a emp s o di e en ia e p ima y om sec-
onda y PIN ha e been published. The e o e, he biologi-
cal signi icance o bo h ypes o PIN emains unknown,
and hei po en ial implica ions o he apeu ic app oach-
es need o be de e mined.
The pu pose o his s udy was o cha ac e ize he ki-
ne ic ea u es o bo h p ima y and seconda y PIN in a se-
ies o su gical specimens. Combined quan i a i e ana-
lyses o p oli e a ion and apop osis ma ke s on issue
sec ions we e pe o med. Ul ima ely, hose pa ame e s
we e o help de ine he kine ic o umo p og ession in
PCa.
This wo k was p esen ed in pa in abs ac o m a he XXII h In-
e na ional Cong ess o he In e na ional Academy o Pa hology,
Nice 1998
M. Koch · S.J. Diaz-Cano
Depa men o Pa hology, GSF-München, Munich, Ge many
M. de Miguel
Depa men o Pa hology, Uni e si y Hospi al “Vi gen Maca ena”,
Se ille, Spain
H. Hö le
Depa men o Pa hology, Technical Uni e si y, Munich, Ge many
S.J. Diaz-Cano
Depa men o Pa hology, S . Ba holomew’s
and he Royal London School o Medicine and Den is y,
London, UK
S.J. Diaz-Cano (✉)
Depa men o Mo bid Ana omy and His opa hology,
The Royal London Hospi al, Whi echapel, London E1 1BB, UK
e-mail: [email p o ec ed].ac.uk
Tel.: +44-171-3777348, Fax: +44-171-3777030
Vi chows A ch (2000) 436:413–420 © Sp inge -Ve lag 2000
ORIGINAL ARTICLE
Michael Koch · Manuel de Miguel · Heinz Hö le
Sal ado J. Diaz-Cano
Kine ic p o iles o in aepi helial and in asi e p os a ic neoplasias:
he key ole o down- egula ed apop osis in umo p og ession
Recei ed: 7 July 1999 / Accep ed: 18 No embe 1999
Ma e ials and me hods
Case selec ion
Fo y consecu i e pa ien s we e included in his s udy, 20 wi h
PIN only (in a backg ound o nodula hype plasia) and 20 wi h co-
exis en low-g ade PCa and PIN in he same specimen. All pa-
ien s showed enla ged p os a e and high se ologic le els o p os-
a e-speci ic an igen (PSA); ei he adical p os a ec omy (all pa-
ien s wi h PCa and 16 wi hou PCa) o ansu e h al esec ion
(4 pa ien s wi hou PCa) was unde aken as app op ia e ea men .
None o hese pa ien s unde wen and ogen dep i a ion he apy
be o e he su gical esec ion, and he ea men was selec ed ac-
co ding o clinicopa hological c i e ia, such as PSA le els o im-
aging analysis. No his ological e idence o ex ap os a ic umo
ex ension was de ec ed, and no pa ien wi h p ima y PIN de el-
oped pa hologically p o en PCa du ing he s udy (mean ollow-up
o 92 mon hs).
The su gical specimens we e ou inely p ocessed and ep esen-
a i e samples we e aken ( ollowing he s anda d c i e ia o sam-
pling in p os a ic esec ions) o es ablish he his ological diagnosis
[24]. Bos wick’s c i e ia we e used o classi y high-g ade PIN [4].
App op ia e a chi al ma e ial was a ailable o addi ional s udies in
each case. The same mic oscopic a eas we e e alua ed wi h e e y
echnique, and a leas i e di e en opog aphic a eas we e
sc eened pe pa ien and pa hologic condi ion (p ima y PIN, PCa,
and seconda y PIN), 50 HPF o he whole oci being e alua ed om
each. All hese sco es we e eco ded and conside ed independen ly
o alida e he esul s pe pa ien and pe pa hologic condi ion.
MIB-1 immunos aining
The sec ions we e moun ed on posi i ely cha ged mic oscope
slides (Supe os Plus, Menzel, Ge many) and baked a 60°C
o 2 h. A e ou ine dewaxing (xylene), ehyd a ion (e hanol)
and endogenous pe oxidase quenching (0.5% H2O2in me hanol,
1–0 min), he sec ions we e mic owa ed in 10 mM ci a e bu e ,
pH 6.0, o 20 min and se ially incuba ed wi h polyclonal ho se
se um (1/100 dilu ion, Dako Denma k) o 20 min, speci ic p ima-
y an ibody (4 µg/ml, Diano a Hambu g, Ge many) o e nigh a
4°C, bio inyla ed an i-mouse an ibody (1/200 dilu ion, Dako Den-
ma k) o 30 min, and pe oxidase-labeled a idin–bio in complex
(1/100 dilu ion, Dako Denma k) o 60 min. All incuba ions we e
pe o med in a mois chambe a oom empe a u e unless o he -
wise speci ied. The eac ion was de eloped unde mic oscopic
con ol, using 3,3’-diaminobenzidine e ahyd ochlo ide as ch o-
mogen (Sigma, S . Louis, Mo.), and he sec ions we e coun e -
s ained wi h hema oxylin.
Bo h posi i e ( eac i e lymph node) and nega i e (omi ing he
p ima y an ibody) con ols we e un simul aneously.
ISEL o DNA agmen s
Since ex ensi e DNA agmen a ion is an impo an cha ac e is ic
o apop osis, isualiza ion o DNA b eaks has p o ed use ul in he
iden i ica ion o apop o ic cells [43]. This ex ensi e DNA ag-
men a ion esul s in a high densi y o 5’-p o uding ends, which
can be de ec ed using he Klenow agmen o DNA polyme ase I
wi h a mix u e o labeled nucleo ides [48]. B ie ly, he sec ions
we e depa a inized and hyd a ed as desc ibed abo e. A e incu-
ba ion in 2×SSC bu e (80°C, 20 min) and p o ein diges ion
(500 µg/ml p onase in 10 mM T is-HCI pH 7.5, 10 mM EDTA,
0.5% SDS, a oom empe a u e o 25 min), he sec ions we e in-
cuba ed wi h he Klenow agmen o E. coli DNA polyme ase I
unde app op ia e condi ions (20 U/ml in 50 mM T is HCI pH 7.5,
10 mM Mg2Cl, 1 mM di hio h ei ol, 250 µg/ml BSA wi h 100 µM
o each dNTP, main aining a p opo ion o 11-digoxigenin-
dUTP/dTTP o 0.35/0.65; 2 h a 37°C). The dig-labeled DNA
agmen s we e immunoenzyma ically de ec ed using an an i-
digoxigenin polyclonal Fab agmen labeled wi h alkaline phos-
pha ase (1/100 dilu ion, Boeh inge -Mannheim, Ge many); he en-
zyma ic eac ion was de eloped unde mic oscopic con ol wi h
ni oblue- e azolium and X-phospha e [12]. The sec ions we e
coun e s ained wi h dilu ed hema oxylin (25%), dehyd a ed, and
moun ed.
Bo h posi i e ( eac i e lymph node) and nega i e (omi ing
DNA polyme ase in he enzyma ic incuba ion) con ols we e si-
mul aneously un.
Quan i ica ion o posi i e nuclei
A leas 50 HPF (50 HPF=7.6 mm2) we e sc eened, o he com-
ple e lesion i smalle (18 pa ien s, 12 wi hou PCa and 6 wi h
PCa), in each pa hologic g oup. Bo h he numbe o posi i e nu-
clei pe HPF and he numbe o neoplas ic duc al cells in e cep ed
by he mic oscope ield diame e we e eco ded. The las sco e
was used o es ima e he numbe o neoplas ic cells pe HPF using
he o mula N=(nπ/4)2, whe e Nis he numbe o es ima ed cells
pe HPF and n, he numbe o cells in e cep ed by he mic oscope
ield diame e [11, 25, 44]. The numbe o posi i e nuclei was al-
ways exp essed pe HPF and pe 1,000 neoplas ic cells. Bo h he
a e age and he s anda d de ia ion (SD) alues we e calcula ed as
ep esen a i e sco es pe pa hologic condi ion and pa ien .
The h eshold o posi i i y was expe imen ally es ablished a
he posi i e con ol in each s aining ba ch. Only hose nuclei
showing s aining ea u es simila o hose o hei co esponding
posi i e con ol we e conside ed posi i e o a gi en ma ke
(MIB-1, ISEL). We only kep , o quan i ica ion, hose nuclei in
he same ocus le el aken as ep esen a i e o any gi en HPF.
Slide cy ome ic analysis o DNA con en
Feulgen-s ained sec ions we e used o DNA quan i ica ion [1],
using he Cell Analysis Sys em (CAS) model 200 and Quan i a i e
DNA Analysis so wa e (Bec on-Dickinson). A leas 200 nuclei
we e e alua ed om each ocus and he esul s eco ded sepa a e-
ly. F om he same slide, bo h lymphocy es and nonneoplas ic duc-
al cells we e used as diploid con ols. Ex e nal diploid con ols
( a hepa ocy es p o ided by Bec on-Dickinson) we e included in
each s aining ba ch o no malize esul s. Se e al 5-µm sec ions
we e used o his analysis, acco ding o p e iously published p o-
ocols ha ha e p o en alid in such ma e ial [19, 29, 46].
Only comple e, nono e lapping and ocused nuclei we e in e -
ac i ely selec ed, beginning in he mos cellula a ea. The his o-
g am o nuclea op ical densi y was used o e alua e he DNA in-
dex (as e e ed o hei co esponding diploid con ols), he p o-
li e a ion a e (PR = S + G2+ M / G1+ S + G2+ M, exp essed as a
pe cen age), and he a io be ween he nuclea a ea and he DNA
con en o he cells in each cell cycle phase. The las a iable was
also e e ed o he co esponding alues in he nonneoplas ic duc-
al cells o he p os a e o no malize he esul s.
S a is ical analysis
The a e age and SD o e e y a iable in each pa hologic condi ion
(p ima y PIN, seconda y PIN, and PCa) we e s a is ically com-
pa ed by means o Fishe ’s exac es s. The da a we e s a i ied in
wo subse s, abo e and below he co esponding h eshold, in each
pa hologic g oup. The h esholds we e 3% o MIB-1 immunoex-
p ession, 0.4% o ISEL o DNA agmen s, and 35 cells/HPF o
cellula i y es ima ion. Di e ences we e conside ed s a is ically
signi ican i P<0.05.
Resul s
Pa ien s ages a ied be ween 51 and 64 yea s (a e age
56) in he g oup wi hou PCa and be ween 60 and
414
71 yea s (a e age 68) in he g oup wi h PCa. All cases
showed u ing o mic opapilla y high-g ade PIN wi h no
e idence o comedonec osis, ega dless o whe he o
no he e was any associa ion wi h PCa. The PCa was
sco ed (Gleason) ≤4 in all pa ien s. The MIB-1 index
showed a p og essi e inc ease om seconda y PIN
h ough p ima y PIN o low-g ade PCa, while he ISEL
index e ealed he lowes sco es in low-g ade PCa and
he highes in seconda y PIN (Table 1).
Combined analysis o kine ic indices in each pa ho-
logic condi ion showed a di ec co ela ion be ween p o-
li e a ion and apop osis in p ima y PIN, while he e was
an in e se co ela ion be ween he wo in bo h low-g ade
PCa and seconda y PIN (Fig. 1). These opposi e pa e ns
we e mainly due o e y low apop osis a es in low-
g ade PCa (in 80% o hese cases ISEL indices we e
lowe han 0.4%) and bo h ela i ely low PR and com-
pa a i ely high ISEL in seconda y PIN (Table 1, Figs. 1,
2). MIB-1 indices below 3% we e obse ed in 86% o
seconda y PIN, and he ISEL index sco ed highe han
0.4% in 80% o hem (Fig. 3). Those pa e ns esul ed in
he highes kine ic ad an age o low-g ade PCa; he ki-
ne ic index (PR-ISEL di e ence) in low-g ade PCa (a -
e age, 3.4%) was 17- old ha in p ima y PIN (a e age,
0.2%) and was highe in bo h hese condi ions (low-
g ade PCa and p ima y PIN) han in seconda y PIN (ki-
ne ic index, –1.8%).
The s a is ical analysis e ealed no signi ican di e -
ences be ween p ima y PIN and low-g ade PCa o ei he
MIB-1 index (P=0.56) o ISEL index (P=0.09). Al-
hough a disc e e inc ease in he MIB-1 index was ob-
se ed in low-g ade PCa (Table 1), he cases we e equal-
ly dis ibu ed a ound (50% below and 50% abo e) 3%
(Fig. 1). Bo h p ima y PIN and low-g ade PCa showed
low ISEL indices, which we e lowe in low-g ade PCa
han in p ima y PIN bu wi h wide in e g oup o e lap-
ping, p ecluding any s a is ical conclusion (Table 1).
Anyway, he p og ession p ima y PIN →low-g ade
PCa was kine ically ela ed o down- egula ion o apop-
osis: 80% o cases o low-g ade PCa showed an ISEL
index ≤0.4%, while only 50% o p ima y PIN e ealed
simila sco es (Fig. 1). Tha endency can only be con-
i med in g oups wi h less a iabili y and wi h mo e
cases included in he analysis.
On he o he hand, seconda y PIN showed s a is ically
signi ican kine ic ea u es om bo h p ima y PIN and
low-g ade PCa (P<0.0001 in all compa isons, o bo h
MIB-1 and ISEL indices). Seconda y PIN was kine ical-
ly cha ac e ized by he lowes MIB-1 index and he high-
es ISEL index (Table 1), he e e se pa e n o low-
g ade PCa (Fig. 1), which was cha ac e ized by low p o-
li e a ion (MIB-1 index ≤3% in 86% o cases) and high
apop osis (ISEL index >0.4% in 80% o cases).
The slide cy ome ic analysis o DNA con en e-
ealed a leas wo G0/G1cells in all cases bu 4 (2 p ima-
y PIN and 2 in asi e low-g ade PCa). The a e age DNA
con en o he p e alen G0/G1peak showed a p og essi e
inc ease om p ima y PIN h ough in asi e low-g ade
PCa o seconda y PIN (Table 2), wi h diploid G0/G1cells
p edomina ing in p ima y PIN and low-g ade PCa and
hype diploid G0/G1cells in seconda y PIN (Fig. 4). Bo h
he nuclea a ea and he nuclea a ea / DNA index a io
e ealed he highes sco e o in asi e low-g ade PCa
and he lowes o p ima y PIN (Table 2). The p esence
o mul iple cases wi h wo G0/G1peaks p ecluded an ap-
p op ia e and eliable e alua ion o p oli e a i e index
om he DNA his og ams in each g oup.
Low-g ade PCa displayed he highes cellula i y sco-
es, whe eas p ima y PIN had he lowes sco es and sec-
onda y PIN e ealed a e age alues be ween hose o p i-
ma y PIN and low-g ade PCa, bu wi h he highes a i-
abili y (Table 1). No di e ences we e de ec ed in a com-
pa ison be ween low-g ade PCa and seconda y PIN. Low-
g ade PCa showed sligh ly highe cellula i y han second-
a y PIN, bu he high a iabili y o he la e esul ed in a
415
Table 1 P oli e a i e and apop o ic ea u es in in aduc al and in-
asi e neoplasms o he p os a e (PIN p os a ic in aepi helial neo-
plasia, PCa p os a ic ca cinoma)
P ima y PIN In asi e PCa Seconda y PIN
(Mean ± SD) (Mean ± SD) (Mean ± SD)
M1B1 index 2.5±2.1 3.5±1.7 1.9±0.7
ISEL index 0.5±0.3 0.1±0.3 3.7±3.3
Cellula i y 26.3±7.1 39.0±8.8 32.9±14.3
Fig. 1 Kine ic p o iles in p os a ic in aepi helial neoplasia (PIN)
and low-g ade p os a ic ca cinoma (PCa). Each ba ep esen s he
pe cen age o cases e ealing a gi en ea u e (p oli e a ion o ap-
op osis). The cases we e ca ego ized in wo g oups o bo h p oli -
e a ion index (MIB-1, h eshold 3%) and apop o ic index (in si u
end-labeling [ISEL], h eshold 4‰). The p og ession om p ima-
y PIN o low-g ade PCa was kine ically de ined by signi ican
down- egula ion o apop osis wi h a mode a e bu no signi ican
inc ease in p oli e a ion. The opposi e pa e n cha ac e ized he
ansi ion p ima y PIN in o seconda y PIN. Bo h p oli e a ion and
apop osis we e signi ican ly di e en in seconda y PIN (Fishe ’s
exac es )
Table 2 Slide cy ome ic analysis o DNA con en in in aduc al
and in asi e neoplasms o he p os a e
P ima y PIN In asi e PCa Seconda y PIN
(Mean ± SD) (Mean ± SD) (Mean ± SD)
Nuclea a ea 42.80±6.34 41.11±4.25
(NuA) 33.04±5.66
DNA index (DI) 1.11±0.16 1.12±0.11 1.22±0.07
NuA / DI 29.74±5.10 38.28±5.66 33.62±3.48
b oad unde ined window p ecluding any s a is ical conclu-
sion. E en a e cellula i y ca ego iza ion, no di e ences
we e de ec ed: 50% o low-g ade PCa and 60% o second-
a y PIN e ealed sco es o ≤35 cells/HPF (P=0.1004).
Howe e , p ima y PIN was p o en s a is ically di e en
om bo h low-g ade PCa and seconda y PIN (P<0.0001
in bo h cases). P ima y PIN showed he lowes cellula i y
o all g oups (Table 1), 80% o hem had ≤35 cells/HPF.
Discussion
Two main in e ences could be d awn in his s udy. Fi s ,
PIN is kine ically di e en acco ding as whe he he e is
coexis en PCa. Secondly, apop osis plays an essen ial
ole in ea ly s eps o p os a e umo igenesis, while p o-
li e a ion seems o be o seconda y impo ance in his
p og ession. We discuss hese poin s below.
Di e en cell kine ic p o iles cha ac e ized PIN asso-
cia ed and no associa ed wi h coexis en PCa. The ela-
i ely low p oli e a ion and ela i ely high apop osis o
PIN wi h coexis en PCa (seconda y PIN) we e demon-
s ably s a is ically di e en om hose o PIN wi h no
PCa (p ima y PIN). This di e en ial kine ic e olu ion
sugges s ha hey ep esen biologically di e en condi-
ions, al hough wi h simila mo phology. High-g ade
PIN is a neoplasm o unce ain biologic beha io , which
occu s as a p ima y disease bu exis s mos o en in asso-
416
Fig. 2 In si u end-labeling in a
p ima y PIN, bseconda y PIN,
and clow-g ade PCa. Ve y low
signal was de ec ed in duc al
cells a ec ed by ei he p ima y
PIN o low-g ade PCa, whe eas
signi ican ly highe alues
we e ob ained in seconda y
PIN. No e he posi i e signal
p o ided by s omal and endo-
helial cells in c(in e nal con-
ol). a,b×400; c×200
cia ion wi h in asi e PCa; i s clinicopa hological co e-
la es emain o be de ined. The his ological and biologi-
cal in o ma ion on high-g ade PIN is based mainly on
case se ies o seconda y PIN, and he e a e no pape s ad-
d essing he kine ic and biologic di e ences be ween
p ima y and seconda y PIN. Ou esul s display wo dis-
inc i e kine ic p o iles o hem. P ima y PIN cases
we e e enly dis ibu ed a ound MIB-1 and ISEL h esh-
olds (50% abo e and 50% below), whe eas a dec easing
PR (86% o cases had an MIB-1 index <3%) and inc eas-
ing ISEL index (80% o cases showed an ISEL index
>0.4%) cha ac e ized seconda y PIN (Figs. 2, 4). The ki-
ne ic pa e n o low-g ade PCa was he opposi e o ha
obse ed in seconda y PIN ( ela i ely high p oli e a ion
and compa a i ely low apop osis) and close o ha o
p ima y PIN han o ha o seconda y PIN. When all
hese ea u es a e conside ed oge he , he mos likely ki-
ne ic e olu ion o seconda y PIN (Fig. 4) seems o be
om p ima y PIN (di e gen p og ession, in asi e and
in aduc al) a he han om low-g ade PCa (linea p o-
g ession, p ima y PIN →low-g ade PCa →seconda y
PIN).
The kine ic ea u es o seconda y PIN a e consis en
wi h an e ol ed umo cell s age wi h eg essi e ea u es
(low p oli e a ion and high apop osis). Ac ually, in si u
gene ic analysis o coexis en PIN and PCa oci o en
shows simila ch omosomal anomalies, al hough se e al
PIN oci ha e been demons a ed o ha e mo e al e a-
ions han ma ched ca cinoma oci [39]. P elimina y e-
sul s om a compa a i e genomic hyb idiza ion analysis
o PIN om his se ies e ealed mo e ex ensi e gene ic
abno mali ies in seconda y PIN and he coexis en PCa
(20q12, 12q14, 9q22, and 16p12) han in p ima y PIN
(20q and 16p) [28]. Those indings sugges ha PIN oci
can unde go mo e ex ensi e ch omosome e olu ion han
417
Fig. 3a, b P ima y PIN. Rela-
i ely low p oli e a ion a e
cha ac e ized PIN, as de ec ed
by nuclea s aining wi h MIB-1
an ibody. aH&E, ×250;
bMIB-1, ×400
Fig. 4 DNA his og am pa e ns in ap ima y and bseconda y
PIN. Diploid G0/G1cells we e equen ly ound in p ima y PIN
and low-g ade PCa, while seconda y PIN e ealed hype diploid
G0/G1cells (peak labeled 1in bo h panels)

PCa and would also suppo a po en ial mul i ocali y in
he e olu ion o PCa [2, 3, 6]. Likewise, hey poin o an
independen clonal e olu ion o seconda y PIN cells, di -
e en om ha o he sequence p ima y PIN →low-
g ade PCa [28]. A p og essi e accumula ion o gene ic
abno mali ies in PIN would inc ease he p obabili y o
gene ically le hal e en s in hose umo cells, hus ising
he apop osis a e. The inc ease in he cell loss de e -
mines a educ ion in he pool o mi o ically ac i e cells
and, he e o e, lowe s PR. Howe e , his up- egula ed
apop osis pa e n is no unique o high-g ade PIN and
has also been desc ibed in duc al ca cinoma in si u
(DCIS) o he b eas . Ha n e al. [21] epo ed signi i-
can ly highe ISEL indices in b eas DCIS han in ei he
in asi e duc al ca cinomas o me as a ic b eas ca cino-
mas, s ongly co ela ed wi h abno mal p53 exp ession
in he in aduc al componen . Mul iple dele ions in ol -
ing se e al umo supp esso genes a e ela i ely e-
quen indings in in aepi helial neoplasms [17, 20, 21,
41]. We he e o e p opose ha a dis inc ion be made be-
ween p ima y and seconda y PIN, in a simila way o
he dis inc ion in e minology ha is widely accep ed in
bladde pa hology [37, 49, 50]. This would mean in e -
p e ing seconda y PIN as pa o he e olu ion o p os-
a e ca cinoma a he han as a p ecu so , as sugges ed in
some clinicopa hological s udies [30, 42].
Ou esul s also emphasize he key ole o apop osis
in ea ly p os a ic neoplasms. Ini ially, he associa ion o
high-g ade PIN wi h low-g ade PCa migh be su p ising,
bu p e ious epo s ha e demons a ed i [38, 40] and
we mus conside ha PIN g ading is based on cy ologi-
cal ea u es while Gleason g ading elies only on a chi-
ec u al pa ame e s and mos PCa show a high nuclea
g ade. The e o e, ha ansi ion is no so su p ising, al-
hough he olume o PIN is posi i ely co ela ed wi h
Gleason sco e [40]. The apop osis analysis, con as ed
by DNA-ploidy and ISEL o agmen ed DNA, showed
ex emely low indices in low-g ade PCa (80% o low-
g ade PCa displayed ISEL index <0.4% and e ealed
he highes nuclea a ea/DNA index a io), smalle
han in p ima y PIN. A he kine ic le el, hese wo con-
di ions we e p o en o be s a is ically di e en om sec-
onda y PIN, essen ially as he esul o apop osis down-
egula ion. Unde physiologic con ol, apop osis closely
co ela es wi h p oli e a ion o main ain a cons an cell
numbe [27]. A con inuous inc ease in p oli e a ion
ma ke s no mally de ines umo p og ession, wi h a pa -
allel inc ease in apop osis [43]. P io o any signi ican
di e ence in p oli e a ion ma ke s, in aepi helial lesions
seem o down- egula e apop osis, esul ing in a kine ic
ad an age and monoclonal expansions speci ically o
hese lesions, as has been shown in b eas DCIS [12], be-
nign ad enal co ical p oli e a i e lesions [8, 13], and ad-
enal medulla y hype plasia [10, 16] o C-cell hype pla-
sias in MEN-2A [9, 14, 51]. Dec eased apop o ic cell
loss would inc ease he cellula pool and allow he accu-
mula ion o gene ically damaged cells, ending in a con-
e gen cellula selec ion. Tha selec ion p ocess would
explain bo h umo p og ession and he e ogenei y,
whe he o no ela ed wi h and ogen le els o wi h an-
d ogen ecep o abno mali ies [26, 32, 34, 35].
P e ious epo s showed a p og essi e and a iable
inc ease o apop osis in PIN and PCa, bu wi h SDs
equal o o highe han he a e age [18], p ecluding any
s a is ical assessmen . This inding has been p oposed as
an indica ion o he con inuum, o pa hway, leading o
PCa. Howe e , apop o ic indices a e no mally low and
equi e sc eening o se e al HPF o achie e eliable e-
sul s. This is especially ue o he e ogeneous condi-
ions, such as PCa. Tha inhe en he e ogenei y de e -
mines g oup o e lapping and makes i di icul (and
some imes impossible) o each s a is ical conclusions.
P oli e a ion pa ame e s ha e been shown o be use ul
in he diagnosis o in aepi helial lesions (dis inc ion o
a ypical hype plasias om ca cinomas in si u) and di-
ec ly co ela ed wi h umo g ading [5, 7, 23, 36]. Ou
p oli e a ion esul s sugges a kine ic di e ence be ween
p ima y and seconda y PIN: only seconda y PIN was
p o en o be s a is ically di e en om bo h p ima y
PIN and low-g ade PCa, wi h highe sco es in he la e
condi ions han in seconda y PIN (Table 1). We ha e
ound a simila kine ic pa e n in CIS associa ed wi h
muscle in asi e ansi ional cell ca cinoma o he u i-
na y bladde : signi ican ly dec eased p oli e a i e indi-
ces we e e ealed in he in aepi helial compa men han
in he in asi e one [17].
Ou esul s a e in he ange o g ade-1 PCa epo ed
by Helpap [23], and bo h his and ou s a e lowe han
hose epo ed by Tamboli e al. [45]. These las au ho s
did no p o ide MIB-1 indices by umo g ade and s age,
bu all hei umo s had Gleason’s sco es o 5+, and o e
one- hi d o hem we e s age III–IV. Tha is a di e en
se ies dis ibu ion om ou s, which showed a combined
Gleason sco e o ≤4. The MIB-1 indices p e iously e-
po ed o PIN a e in he ange o ou sco es in p ima y
PIN bu e eal high a iabili y ( he SD was 1.5- old he
co esponding a e age) [45], p obably an exp ession o
umo he e ogenei y. Bo h in a umo al he e ogenei y
and he quan i a ion me hod will con ibu e o hese dis-
c epan esul s. We sc eened 50 HPF/ ocus (o he com-
ple e lesion i smalle ) and 5 di e en oci pe pa ien
and pa hologic condi ion o dec ease he sco e a iabili y
( he smalle he SD he highe he measu emen accu a-
cy) and o ake he in a umo al he e ogenei y in o con-
side a ion. Ou SD alues we e always below a e age,
sugges ing he use o a be e sampling me hod o umo
e alua ion han o p e iously epo ed esul s [23, 45].
S udies in o he o gans ha e demons a ed a opog aphic
he e ogenei y o kine ic ea u es missed wi h he s an-
da d sc eening me hods [11, 16].
In summa y, wo kine ic p o iles could be d awn in
PIN, depending on whe he o no i was associa ed wi h
coexis en low-g ade PCa, which sugges s ha hese e-
p esen biologically di e en condi ions. The unique ki-
ne ic p o ile o seconda y PIN, wi h ela i ely low p o-
li e a ion and ela i ely high apop osis, is consis en wi h
an e ol ed umo cell s age wi h eg essi e ea u es a h-
e han wi h a p ecu so lesion. Ea ly s eps in umo igen-
418
esis in low-g ade p os a e ca cinomas a e kine ically
cha ac e ized by down- egula ed apop osis a he han by
inc eased p oli e a ion.
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