Kinetic profiles of intraepithelial and invasive prostatic neoplasias: the key role of down-regulated apoptosis in tumor progression

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. 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