Nucleolar organiser regions (AgNORS) in anal intraepithelial neoplasia and invasive anal

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1 J Clin Pthol 1992;5: Deprtment of Surgery, Clinicl Sciences Centre, Northern Generl Hospitl, Sheffield S5 7AU O A Ogunbiyi J H Scholefield K Rogers Deprtment of Obstetrics nd Gynecology F Shrp R Ginsberg Correspondence to: Mr A Ogunbiyi Accepted for publiction 6April 1992 Nucleolr orgniser regions (AgNORS) in nl intrepithelil neoplsi nd invsive nl squmous cell crcinom A Ogunbiyi, J H Scholefield, F Shrp, R Ginsberg, K Rogers Abstrct Aim: To evlute the usefulness of counting nucleolr orgniser region ssocited proteins (AgNORs) in the mngement of nl squmous neoplsi. Method: Using silver stining technique for NOR ssocited proteins, 32 routinely processed prffin wx embedded sections of nl epithelium were ssessed. These consisted of norml nl epithelium (n = 9), nl intrepithelil neoplsi (AIN) grdes I (n = 5), nd III (n = 13), nd invsive squmous neoplsi of the nus (n = 5). Results: The medin AgNOR counts for every 1 cells re s follows: norml nl epithelium 215 (95% CI ); AIN I 321 (95% CI 28971); AIN III 32 (95% CI 81); nd invsive squmous cell crcinom of the nus 5 51 (95% CI ). There were significnt differences between AgNOR counts in nl cncer nd norml epithelium (p < 5; MnnWhitney U test)), AIN III nd norml nl epithelium (p <.5), nd AIN III nd AIN I (p <.5). No significnt differences were observed between AIN I nd norml nl epithelium, nl cncer nd AIN I, nd nl cncer nd AIN III. There ws considerble degree of overlp mong the different groups. Conclusions: Despite the strong ssocition between AgNOR vlues nd degree of dysplsi, the vribility within pthologicl grde my preclude the doption of this technique on its own s prognostic indictor. It my, however, be useful in conjunction with other mrkers of neoplstic growth such s cmyc oncogene mplifiction or overexpression s mrker of disese progression in AIN nd invsive nl squmous cell cncer. (7 Clin Pthol 1992;5:889893) Squmous cell crcinom of the nus is n uncommon tumour, comprising 3% of lrge bowel tumours.' However, in some groups such s immunosuppressed orgn trnsplnt recipients, homosexul men who prctise receptive nl intercourse, nd in group IV (Centers for Disese Control) HIV seropositive persons, the incidence of squmous nl cncer is incresing.2 Anl intrepithelil neoplsi (AIN), which ws first described by Fenger nd Nielsen in nd which ws thought initilly to be rre, hs lso been 889 shown to be incresing in prevlence in the bove groups.`6 Although the prevlence nd clinicl importnce of AIN remins unknown, it hs been suggested tht there my be possible prllel between AIN nd cervicl intrepithelil neoplsi (CIN) s regrds progression to invsive squmous cncer. If this is true, the vilbility of mrker identifying those AIN lesions most likely to progress to invsive cncer would be extremely useful in the mngement of ptients who re thought to be t risk. Nucleolr orgniser regions (NORs) re loops of ribosoml DNA (rdna) occurring in the nucleoli of cells, which trnscribe to ribosoml RNA (rrna) nd ultimtely direct ribosome nd protein formtion.7 Becuse of the close ssocition between NORs nd cell ctivity, their number or size is thought to reflect cell prolifertion, trnsformtion, nd even mlignncy. NORs re redily identified by mens of the silver binding (rgyrophilli) of their ssocited proteinsthe AgNOR technique.8the method hs permitted the recognition of NORs in chromosome spreds, whole cells, nd histologicl sections. Using the AgNOR technique on routinely processed prffin wx embedded tissue, there hve been reports tht it is useful in discriminting between benign melnonevi nd mlignnt melnom,9 Spitz nd pigmented spindle cell nevi from melnom,1 nd between low nd high grde lymphoms." Significnt differences in the AgNOR counts of the different grdes of CIN hve been shown recently,'2 lthough the degree of overlp between the grdes limits the use of this method in the dignosis. This study is, to our knowledge, the first systemtic study of AgNORs in AIN nd invsive nl cncer. Using silver stining technique for NOR ssocited proteins, we hve ttempted to identify mrker which could be useful in the identifiction of ptients with AIN t risk of progression to invsive nl cncer, nd hence their subsequent mngement. Methods Thirty two nl epithelil specimens were studied. These comprised: nine norml nl epithelium, five AIN I, 13 AIN III nd five squmous cncers. These were tken from the rchivl files of the Histopthology Deprtment t the Northern Generl Hospitl, Sheffield. The histology on ll specimens ws reviewed by one consultnt pthologist with n

2 89 Ogunbiyi, Scholefield, Shrp, Ginsberg, Rogers &,k:.&n. V.mft.S..: :11. 1,. ' M. mil,... p V: Alm.. f: I.xp T'.1..,.. A..1:U. 'm wpb ' :.,::,:.. AAAL.:._ i W 1 N ".t described interest in nogenitl neoplsi. The specimens hd been fixed in 1% buffered formlinphosphte nd prffin wx embedded. Sections (5 gum thick) were dewxed in xylene nd hydrted through grded ethnols to deionised wter. Adjcent sections from ech block were stined with hemtoxylin nd eosin nd for silver binding NOR ssocited protein, s by Smith nd Crocker.'3 The AgNOR stining solution ws prepred by dissolving 2% solution of geltin in 1% formic cid. This solution ws then mixed in rtio of 1:2 with 5% queous silver nitrte. *^ ^ The tissue sections were immersed in the finl working solution nd left for 3 minutes t room temperture in drk room. The slides were then fixed, wshed in distilled wter, w dehydrted in scending grdes of ethnol nd mounted. The presence of NORs is indicted * W by the ppernce of blck silver grnule(s) in * * the nucleus (fig 1). v ;AgNORs present in the nuclei of 1 consecutive norml or typicl nl epithelil cells were counted t mgnifiction of x 1 using n oil immersion lens by single observer. Two methods of AgNOR enumertion were evluted by two observers in preliminry series of 1 specimens. In the first * method the ctul numbers of AgNORs were quntified by counting ech pprent dot even when they touched or overlpped. The interobserver vrition ws 253% using this method. The second method involved counting the number of seprble blck dots in ech nucleus. Overlpping dots were counted s one. Using this method, interobserver vrition ws 58%. The second method ofagnor enumertion ws therefore felt to be more relible nd used for the purpose of this study. The number of nuclei to be counted in ech * :.. A A.P..,..K. ":,::: IV.. 'k..lg:. '9.:.:.. i"n I q.,f specimen ws determined by the cumultive mens technique. Using this procedure, it ws shown tht the men number of AgNORs would not hve been ltered in ny lesion by counting more thn 1 nuclei. All the dt were nlysed using the Mnn Whimey U test for nonprmetric dt. Results For ech specimen, the cumultive mens technique ws used to determine the number of nuclei tht needed to be counted before the mens becme stble. The men ws rbitrrily defined s being stble when the men reched nd styed within ± 5% of the finl men (n = 1). Figure 2 illustrtes moving verge plots f of typicl exmples of norml nl epithelium, AIN III, nd invsive squmous crcinom of the nus. In the illustrted exmples the men vlues becme stble fter 3 nuclei hd been counted for norml nl epithelium nd 5 nuclei for both AIN3 nd nl squmous cncer. The medin numbers of nuclei counted in ech group before the men becme stble re s follows: norml nl epithelium (n = 9) 35 (rnge 27); AIN I (n = 5) (rnge 37); AIN III (n = 13) 5 (rnge 37); Figure 1 AgNORs in (A) norml nl epithelium nd (B) AIN I. The AgN'ORs show nd invsive nl squmous cell crcinom up s single or multiple blck dots in the nucleus (shown by rrows). (n = 5) 5 (rnge 27).

3 .>... : : : AgNORS in nl intrepithelil neoplsi nd invsive nl squmous cell crcinom * *; VW W., : *~~~~ql.* F S.:. * Ib 1 * V :. s "., A_ VIC"" *::_6.1 6 A S : 9 W. ' V to.*.. * A. i t _~~q o.111mi! IF. *:..A b:. : % &.Or. ti. tk. 6 : r :: Vb!: sob, % / ~ ~ ~ ~ ~ B; ::. :X :.: * * * it :*. :b..^ w._.t:..t. t...: x,. ss t :. *. S', *B,: :....^.:_w.s I ::, i.i : lk::.ii: f qw 1 Figure 1 continued AgNORs in (C) AIN III nd (D) invsive nl squmous crcinom. The AgNORs show up s single or multiple blck dots in the nucleus (shown by rrows). The dots in AIN III nd nl cncer re smller nd form numerous clusters compred with norml nl epithelium nd AIN I. The scttergrm in fig 3 depicts the men AgNOR counts for ech specimen in ll four dignostic groups. In ll specimens AgNORs t*w were clerly visible s blck dots of vrying sizes in the nuclei. These were rrnged into one or more clusters or s individul stellitesextrnuceolr. In AIN III nd invsive nl squmous cncer specimens there were more AgNOR clusters s well s extrnucleolr *# AgNORs present compred with norml nl epithelium nd AIN I. The finl medin vlues for ech group re shown in tble 1. Although there is progressive increse in the medin AgNOR vlues, there is considerble degree of overlp between the four groups s illus * trted in the scttergrm. Using the Mnn Whitney U test for nonprmetric dt (tble 2), there were significnt differences between 1* AgNOR counts in nl cncer nd norml nl epithelium (p < 5), AIN III nd norml nl epithelium (p < 5), nd AIN III nd AIN I (p < 5). No significnt differences were observed between AIN I nd norml nl epithelium, nl cncer nd AIN I, nd cncer nd AIN III. Discussion Although possible prllels between AIN nd CIN hve been suggested,6 the nturl history of AIN is still unknown. However, AIN III hs been detected in resection specimens for squmous nl cncer. 1 These lesions were usully found djcent to the tumours s well s in res seprted from the tumour by * norml mucos. AIN seems to be firly common in certin t risk groups, nd in view of the rrity of nl crcinom (3% of bowel ** cncers), most of the AIN lesions seem to regress or remin sttic. This poses problems in the mngement of AIN lesions, s treting ll lesions would result in unnecessry tretment of lrge numbers of ptients. On the other hnd, the possibility tht smll number of ptients with untreted AIN my progress to invsive cncer hs to be considered. How, then, do we determine which AIN lesions re likely to progress to invsive cncer? Idelly mrker identifying bnorml nl cells would id in the dignosis nd follow up of ptients with MIN. *t ti*e This study gives the first description of AgNOR counts in AIN nd invsive nl squmous cncer. We hve tried to evlute the usefulness of AgNORs in differentiting between nl neoplstic lesions nd hence the possibility of using the method s mrker in following the progress of AN lesions. The AgNOR technique hs been used by cytogeneticists for over decde, lthough it hs only recently been pplied in histopthology." The Tble 1 Medin AgNOR vlues Medin Norml % (18939) AIN I % (28971) AIN III 32 95% (8 1) Crcinom % (28162) CI 891

4 892 Ogunbiyi, Scholefield, Shrp, Ginsberg, Rogers en U, c z i 6 Anl Ploton et l (1986) were the first to suggest tht crcinom interphse AgNOR counts could indicte 5 mlignnt disese when they showed tht i_ AgNOR counts in prosttic crcinom gretly IITNIII AI ~ exceeded tht in benign prosttic hyperpl AINi. 1 s \_ + Since then there hve been severl studies / describing the ppliction of silver stining 3 methods for the demonstrtion of AgNORs in histologicl mteril from wide rnge of Norml 2 Norml diseses. These studies hve shown tht mlignnt cells cn be distinguished from corresponding benign or norml cells on the bsis of 1 higher quntity of interphse AgNORs. 9 2 Our results hve shown tht lthough there o2,,,, is progressive increse in the medin number o so 9 1 of AgNOR counts in norml, AIN I, AIN III Nuclei counted nd invsive cncer, there is high degree of overlp between the groups, prticulrly re 2 Cumultive mens technique: moving verge plots of typicl exmples of between AIN III nd invsive cncer. For Ftgu nornnl nl epithelium, AIN III nd invsive nl squmous cell crcinom. The vertzicl brs indicte the number of nuclei counted to chieve stble men C 'S z 3 prcticl purposes, therefore, AgNOR counts re of limited vlue in discriminting between grdes of AIN nd invsive cncer nd re unlikely to be suitble s mrker of disese 1 ~ progression. These results re similr to those 13 x obtined by Drne et l,21 who evluted 2 AgNORs in norml endocervix, denocrci 11 x nom in situ (AIS), nd invsive denocrci x nom of the cervix. They found tht lthough 9 x X AgNOR counts differentited between norml 8 x endocervicl cells on the one hnd nd AIS 7 x x nd invsive denocrcinom on the other, 6 Xx X x between there ws cses of significnt AIS nd invsive degree denocrci of overlp X nom. This suggested tht lthough AIS ws 3X 5t potentil premlignnt precursor of invsive x x denocrcinom, the ssessment of AgNORs 2 ws of limited use in discriminting between 1 the histologicl types of cervicl crcinom. O The evlution of AgNORs in CIN hs lso Norml AIN AIN III Anl 2 crcinom been shown to be of limited dignostic vlue.'2 In tht study the men number of AgNORs Figutre 3 Scttergrm to show the distribution of totl numbers ofagnors per 1 cells ws shown to increse stedily in the three of ezch cse exmined. The horizontl brs indicte the medin for ech group. grdes of CIN with some significnt differences between the groups. However, there ws overlp between the grdes, thereby mking NORs re present on the short rms of five this method of limited dignostic vlue. chromosomes 13, 1, 15, 21 nd 22 in mn, Recently, studies of oncogene expression in nd hve llowed vrious genetic defects in cervicl intrepithelil neoplsi (CIN) nd metphse chromosome spreds to be n invsive cncer of the cervix hve shown lysed. The nture of the silver stining NOR moleculr ltertions of cmyc oncogene in ssocited proteins shown by the AgNOR crcinom of the cervix s well s overtechnique is not fully known, lthough it is expression of its protein product p thought they my be relted to proteins such s Crook et l2 hve lso demonstrted similr RNA polymerse I,15 C23 (nucleolin), nd chnges in nl squmous cell crcinom, B23 protein.'67 The exct function of these lthough no chnges were observed in the five proteins is uncertin, lthough it is thought specimens of AIN III exmined. Cmyc oncotht they my hve some regultory function in gene expression my serve s mrker of Tble 2 controlling the trnscription of the genes for disese progression, nd studies into this re ribosoml RNA nd hence protein synthesis. currently being undertken in this unit. Sttisticl nlysis ofagnor counts using MnnWhitney U test Point estimte CI P vlue Norml v AlN I ( 291, 931) > 5 Norml v AIN III ( 571, 135) < 5 Norml v nl crcinom ( 619, 329) < 5 AIN I v AIN III ( 5399, 22) < 5 AIN I v nl crcinom ( 71, 1632) > 5 AIN III v nl crcinom 6 95 ( 298, 3679) > 5 1 Morson BC. The pthology nd results of tretment of squmous cell crcinom of the nl cnl nd nl mrgin. Proc Roy Soc Med 196;53: Wexner SD, Milsom JW, Diley TH. The demogrphics of nl cncers re chnging: Identifiction of high risk popultion. Dis Colon Rectum 1987;3: Penn I. Cncers of the nogenitl region in renl trnsplnt recipients. Anlysis of 65 cses. Cncer 1986;58: Plefsky JM, Gonzles J, Greenbltt RM, Ahn DK, Hollnder H. Anl intrepithelil neoplsi nd nl ppillomvirus infection mong homosexul mles with Group IV HIV disese. JAMA 199;263:

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