J Clin Pathol 1990;43:657-660

Nucleolar organiser regions in adenocarcinoma insitu and invasive adenocarcinoma of the cervix

J F Dame, S V Polacarz, E Sheridan, D Anderson, R Ginsberg, F Sharp

AbstractSilver binding nucleolar regions(AgNORs) were evaluated in normalendocervix, adenocarcinoma, and itspotential precursor, adenocarcinoma insitu (AIS), in an attempt to increase anunderstanding of the natural history ofcervical adenocarcinoma and to identifya marker for abnormal endocervical(atypical glandular) cells which couldaid diagnosis and follow up of endo-cervical lesions. For every 50 cells themean AgNOR counts were as follows:normal endocervical cells (n = 15) 79-8(95% Cl 68-91); AIS (n = 20) 200-7 (95%Cl 182-219); and invasive adenocarcin-oma (n = 30) 299 (271-328). There was nooverlap between the groups of normalendocervical cells and invasive aden-ocarcinoma, but there was significantoverlap between cases of invasive aden-ocarcinoma and carcinoma in situ. In sixout of 17 cases with AIS, NOR count inadjacent morphologically normal glan-dular cells ("internal" controls) wasincreased when compared with the"external" (normal endocervical) con-trol group. This suggests the presence ofwider field changes not previously iden-tified using routine histological methods.The findings suggest that AIS is a

potential premalignant precursor ofinvasive adenocarcinoma, but that as-sessment of NORs is of no practical usein discriminating between the his-tological types of cervical carcinoma.

The absolute incidence of adenocarcinoma ofthe cervix seems to have increased in youngwomen over the past decade.l" An increase inthe proportion of adenocarcinomas in series ofcervical carcinomas has also been reported.5 6The natural history of cervical adenocarcin-

oma has been less well investigated than thatof its squamous counterpart. Teshima et alsuggested that adenocarcinoma could arise denovo from "normal" epithelium without pass-ing through an adenocarcinoma in situ (AIS)phase7; this could be explained by the under-diagnosis of AIS."9 The premalignant poten-tial of certain lesions of the endocervixhowever-namely, cervical glandular atypia(CGA) and AIS-remains unclear, and theirdiagnosis and follow up remain difficult.'0

Nucleolar organiser regions (NORs) havean essential role in the transcription of nucleicacid to protein. An apparent increase of

visible NORs in a cell population seems toreflect increased transcriptional activity. NORcounts, visualised as dots using an argyro-philic technique, have been reported to helpin the distinction between high and low gradelymphoma" and benign and malignant melan-omas.'2 A significant difference in the numberof NORs has more recently been reportedwhen comparing benign and malignantstomach lesions, but there was some overlapbetween the two groups."3 The mean numberof NORs has also been shown to increasesteadily in cervical intraepithelial neoplasia(CIN) from grades I to III."4The aims of this study were to use a simple

silver staining technique for NOR protein toinvestigate the association between normalcolumnar epithelial cells of the endocervix,potential malignant precursors, and invasiveadenocarcinoma of the cervix; and to identifya marker for abnormal endocervical (atypicalglandular) cells which might enhance thediagnosis and follow up of endocervicallesions.

MethodsArchival, formalin fixed, and paraffin waxembedded tissue blocks were used. It wasunlikely that any single centre would have hadsufficient material in its histopathologicalarchives to supply the number of specimens ofAIS required: it was therefore essential tocollect the material on a multicentre basis.

Tissue blocks from 30 women with invasivecervical adenocarcinoma and 20 with AIS(cones or punch biopsy specimens) were selec-ted. "External" controls consisted of normalendocervical blocks of tissue obtained from 15patients after total hysterectomy for dysfunc-tional uterine bleeding. All tissues studied hadbeen previously diagnosed using routinehaematoxylin and eosin staining.

Sections from the original blocks were cutat 4 pm, placed on glass slides, dewaxed andrehydrated. Adjacent sections from each blockwere stained with haematoxylin and eosin,and for argyrophilic protein-linked NOR(AgNOR), as described elsewhere.'5 Briefly,all solutions used were freshly prepared usingglass distilled water, and a gelatin (2 g/dl) andformic acid (1 ml/dl) solution was vortexedwith a 500'o solution of silver nitrate (1:2volumes). Staining with this mixture was star-ted shortly after mixing and carried out for 35minutes at room temperature in a darkroom.The slides were then fixed, washed,dehydrated and mounted. The final reaction

Clinical SciencesCentre, NorthernGeneral Hospital,Harries Road,Sheffield S5 7AUJ F Dame,E SheridanD AndersonR GinsbergF SharpDepartment ofPathologyS V PolacarzCorrespondence to:Dr J F DameAccepted for publication15 February 1990

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Figure I "External" normal endocervical control. Most cells contain one or two visibleblack NOR granules (black argyrophilic dots).

product, which indicates the presence of anNOR, is the appearance of a black silvergranule(s) in the nucleus.Using oil immersion (of a final magnifica-

tion of 1000) "total" AgNORs present werecounted in the nuclei of 50 endocervical glan-dular consecutive normal or atypical cells."Internal controls" were also evaluated in theAIS subgroup-that is, morphologically nor-mal columnar epithelium adjacent to AIS.

Results"EXTERNAL" NORMAL ENDOCERVICAL CONTROLCELLSMost cells contained one or two NORs whichtended to be large, with regular outline, and

Figure 2 Scattergram toshow distribution of totalnumbers ofNORs for 50cells of each caseexamined. Horizontal barsindicate the median foreach group.

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evenly stained (fig 1). The median for 15patients studied was 70 NORs/50 cells (950oCl-68 to 91) (fig 2).

AISThe median count for this group of 20patients was 209 NORs/50 cells (95% Cl 182to 219). This showed a significant increase inthe total number ofNORs in this group whencompared with those of the normal controls(Mann-Whitney U test, p < 0-001). Figure 2shows a clear distinction between the externalcontrol and the AIS groups. Furthermore,individual NORs were smaller, occurring inclusters, and more widespread within thenuclei (fig 3).

Adjacent, morphologically normal glan-dular cells ("intemal" controls) in the sectionsof AIS were identified and total number ofNORs counted. This was possible in only 17out of 20 cases. The median count was 98NORs/50 cells (95% Cl 92 to 135), significan-tly higher than the normal external controls.(Mann-Whitney U tests-p < 0O01). Anoticeable increase in NOR count in the inter-nal controls, however was seen in six out of 17patients. The counts in the other groups werewithin external control limits.

INVASIVE ADENOCARCINOMAA significant increase in the NOR count wasseen in the 30 cases of invasive adenocarcin-oma compared with the AIS group (overallmedian 299 NORs/50 cells; p < 0-001) therewas, however, considerable overlap betweenthese two tissue populations (fig 2). Theabnormal features of individual NORs in theinvasive adenocarcinoma sections were as des-cribed for the AIS group, or more pronoun-ced in some cases.

30 Discussiong99 The potential malignant role of AIS has76 received scant attention recently. Rollanson

et al investigated the staining patterns of six* cases of AIS with antiepithelial membrane* antigen (anti-EMA) as part of a larger study

and found that AIS and early adenocarcinoma% stained equally intensely.'6 Similarly, 13 out* of 15 cases of cervical glandular atypia showedJL abnormal dense cytoplasmic and cuticular_~g reactivity with human milk fat globulin/mon-$ oclonal antibody.'7 Normal endocervix

showed only dense cuticular reactivity.Other markers have also been studied:

intermediate filaments,'8 carcinoembryonicantigens, and fat globule membrane antigen.'8Our study is, as far as we know, the firstcontrolled investigation of both potentialprecursors and invasive disease. The numbersof NORs were significantly increased in bothstudy groups when compared with normal

8 columnar epithelium, thus corroborating apotential premalignant role for AIS. This fur-ther confirms the retrospective finding ofglandular atypia in smears taken up to sixyears previously in six out of 13 cases ofinvasive adenocarcinoma"' and histological

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Value ofNOR counts in AIS and invasiveadenocarcinoma6s. - p t- -_F

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Figure 3 Adenocarcinoma in situ: most cells contain numerous visible black NO)granules.

abnormality in five out of 18 similar case

The conventional management of Athe margins of excision are clear of dysjepithelium on cone biopsy, is essentiallyterm colposcopic and longer term cytolfollow-up. The recurrence of AIS is litinitial lesion, likely to be small in sizconfined to a small area of the cervix,routine cytology is known to be poor atting exfoliated atypical glandular cells.2use of an NOR count as a marker peculAIS (and invasive lesions) with addiendocervical sampling, which has re

been shown to be effective in predresidual disease in incompletely e:

squamous cervical intraepithelial neoplcould improve the follow up of these leAfter observing a significantly increasedcount in dysplastic endocervical celhlstudy is being extended to address this isSimple hysterectomy has been suggesi

the ideal surgical management of AlAhalysis of recent data from three UKingdom cancer registries has shoNthreefold increase in cervical adenocarciin 20 to 34 year olds during the 10studied.3 There must be a parallel incre:AIS in the same age group. Hysterectomost undesirable in such young womer

diagnostic cone biopsy is thought ttherapeutic if the margins of excision-areWe observed an increase in NOR cou

morphologically normal endocervical eelium adjacent to AIS in six out of 17Using the c-myc oncogene mono(

antibody P62, similarly abnormal gene exsion or amplification was observed in ninof 14 cases in otherwise normal glands adjto AIS (personal observations, Polacar2Dame JF, Sharp F). These observations ia more widespread field change and suthat simple cone biopsy, even if margiexcision are clear, might be suboptimalment in some cases.The presence of invasion clearly has in

tant therapeutic and prognostic implicaAlthough controversial and extremely dii

IV to recognise,24 probably because of inadequateexperience, early invasion by AIS has beenclearly described.725 Unfortunately, assess-ment of NORs, due the considerable overlap

v between AIS and invasive adenocarcinomagroups in our studies, was a poor discriminantbetween the two conditions. Morphometricanalysis has also shown an unacceptably high

* degree of overlap between them,26 but similar* to squamous cervical intraepithelial neo-

plasia,'4 the median number ofNORs increased0 steadily from AIS to invasive disease. Assess-

t ment of NORs, however, has already beenshown to be of no practical use in discriminat-

4 ing between the histological types of cervicalcarcinoma2 but is thought to be of possible

* prognostic value in breast cancer.28This study adds further support to the

potential premalignant role that AIS has ininvasive adenocarcinoma. It has also identified

R a potential cytological marker for endocervicallesions although assessment ofNORs will be of

9 no practical use for enhancing the diagnosis ofIS, if early stromal invasion.

lasticWe thank Dr M C Anderson (The Samaritan Hospital,

short London), Dr A B Maclean (Hospital for Mothers, Glasgow),Logical Mr J M Monaghan (Oncology Centre, Gateshead) and Mr I

Scott (Derby City Hospital, Derby) for providing some of theke the archival specimens used in this study.e andI20 butdetec-The 1 Dallenbach-Hellweg G. On the origina and histological

liar to structure of adenocarcinoma of endocervix in women

itional under 50 years of age. Path Res Pract 1984;179:38-50.2 Ireland D, Hardiman P, Monaghan JM. Adenocarcinoma of

cently the uterine cervix: a study of 73 cases. Obstet Gynecollicting 1985;65:82-5.

cs3 Chilvers C, Mant D, Pike MC. Cervical adenocarcinoma and

xcised oral contraceptives. Br Med J 1987;295:1446-7.lasia 22 4 Vesterinen E, Forss M, Nieninen U. Increase of cervicalasia, adenocarcinoma: a report of 520 cases of cervical carcin-sions. oma including 112 tumours with glandular elements.NOR Gynecol Oncol 1989;33:49-53.

5 Davis RJ, Moon LB. Increased incidence ofadenocarcinomaS this of uterine cervix. Obstet Gynecol 1975;45:79-83.sue. 6 Gallup DG, Abell MR. Invasive adenocarcinoma of the

* uterine cervix. Obstet Gynecol 1977-49:596-602.ted as 7 Teshima S, Shimosato Y, Kishi K, Ohmi K, Uei Y. Early[S.2023 stage adenocarcinoma of the uterine cervix. Cancer

1985;56: 167-72.Jnited 8 Christopherson WM, Nealon N, Gray LA. Noninvasive

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GW. Adenocarcinoma in situ of the cervix: an underdiag-ase in nosed lesion. Cancer 1981;48:768-73.

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phomas. J Pathol 1987;151:111-8.clear. 12 Crocker J, Silbeck N. Nucleolar organiser region associatednt in protein in cutaneous melanotic lesions: a quantitative

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associated proteins in breast malignancy. HistopatholZ SV, 1988;12:113-25.imply 16 Rollanson TP,' Byrne P, Williams A, Brown G. Expression

of epithelial membrane and 3-fucosyl-N-acetyl-lac-iggest tosamine antigens in cervix uteri with particular referencens of to adenocarcinoma in situ. J Clin Pathol 1988;41:547-52.

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cervical glandular atypia. J Pathol 1987;151:203-8.18 Dabbs DJ, Geisinger KR, Norris HT. Intermediate

npor- filaments in endometrial and endocervical carcinomas. Amtions. J Surg Pathol 1986;10:568-76.

* 19 Boddington MM, Spriggs AI, Cowdell RH. Adenocarcin-fficult oma of the uterine cervix: cytological evidence of a long

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22 Husseinzadek N, Shbaro I, Wesseler T. Predictive value ofcone margins and post-cone endocervical curettage withresidual disease in subsequent hysterectomy. GynecolOncol 1989;33:198-200.

23 Quazilbash AH. In-situ and micro-invasive adenocarcinomaof the uterine cervix. Am J Clin Pathol 1975;64:155-70.

24 Burghardt E. Microinvasive carcinoma in gynaecological

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