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J Clin Pathol 1992;45:889-893

Nucleolar organiser regions (AgNORS) in analintraepithelial neoplasia and invasive analsquamous cell carcinoma

0 A Ogunbiyi, J H Scholefield, F Sharp, R Ginsberg, K Rogers

AbstractAim: To evaluate the usefulness of count-ing nucleolar organiser region associatedproteins (AgNORs) in the management ofanal squamous neoplasia.Method: Using a silver staining techniquefor NOR associated proteins, 32 routinelyprocessed paraffin wax embedded sec-tions of anal epithelium were assessed.These consisted of normal anal epithe-lium (n = 9), anal intraepithelial neoplasia(AIN) grades I (n = 5), and III (n = 13), andinvasive squamous neoplasia of the anus(n = 5).Results: The median AgNOR counts forevery 100 cells are as follows: normal analepithelium 2-15 (95% CI 1.89-3.94); AIN I3-21 (95% CI 2-89-7-14); AIN III 4-32 (95%CI 4-00-8-10); and invasive squamous cellcarcinoma of the anus 5 51 (95% CI2.48-10.62). There were significant differ-ences between AgNOR counts in analcancer and normal epithelium (p < 0 05;Mann-Whitney U test)), AIN III and nor-mal anal epithelium (p < 0.005), and AINIII and AIN I (p < 0.05). No significantdifferences were observed between AIN Iand normal anal epithelium, anal cancerand AIN I, and anal cancer and AIN III.There was a considerable degree of over-lap among the different groups.Conclusions: Despite the strong associa-tion between AgNOR values and degree ofdysplasia, the variability within patho-logical grade may preclude the adoptionof this technique on its own as a prog-nostic indicator. It may, however, be use-ful in conjunction with other markers ofneoplastic growth such as c-myc oncogeneamplification or overexpression as amarker of disease progression in AIN andinvasive anal squamous cell cancer.

Department ofSurgery, ClinicalSciences Centre,Northern GeneralHospital, SheffieldS5 7AUO A OgunbiyiJ H ScholefieldK RogersDepartment ofObstetrics andGynaecologyF SharpR GinsbergCorrespondence to:Mr 0 A OgunbiyiAccepted for publication6April 1992

(7 Clin Pathol 1992;45:889-893)

Squamous cell carcinoma of the anus is an

uncommon tumour, comprising 3% of largebowel tumours.' However, in some groupssuch as immunosuppressed organ transplantrecipients, homosexual men who practisereceptive anal intercourse, and in group IV(Centers for Disease Control) HIV seroposi-tive persons, the incidence of squamous analcancer is increasing.2 4 Anal intraepithelialneoplasia (AIN), which was first described byFenger and Nielsen in 19815 and which was

thought initially to be rare, has also been

shown to be increasing in prevalence in theabove groups.4`6 Although the prevalence andclinical importance ofAIN remains unknown,it has been suggested that there may be apossible parallel between AIN and cervicalintraepithelial neoplasia (CIN) as regards pro-gression to invasive squamous cancer. If this istrue, the availability of a marker identifyingthose AIN lesions most likely to progress toinvasive cancer would be extremely useful inthe management of patients who are thoughtto be at risk.

Nucleolar organiser regions (NORs) areloops of ribosomal DNA (rDNA) occurring inthe nucleoli of cells, which transcribe toribosomal RNA (rRNA) and ultimately directribosome and protein formation.7 Because ofthe close association between NORs and cellactivity, their number or size is thought toreflect cell proliferation, transformation, andeven malignancy. NORs are readily identifiedby means of the silver binding (argyrophillia)of their associated proteins-the AgNOR tech-nique.8The method has permitted the recogni-tion of NORs in chromosome spreads, wholecells, and histological sections. Using theAgNOR technique on routinely processedparaffin wax embedded tissue, there have beenreports that it is useful in discriminatingbetween benign melanonaevi and malignantmelanoma,9 Spitz and pigmented spindle cellnaevi from melanoma,10 and between low andhigh grade lymphomas." Significant differ-ences in the AgNOR counts of the differentgrades of CIN have been shown recently,'2although the degree of overlap between thegrades limits the use of this method in thediagnosis.

This study is, to our knowledge, the firstsystematic study of AgNORs in AIN andinvasive anal cancer. Using a silver stainingtechnique for NOR associated proteins, wehave attempted to identify a marker whichcould be useful in the identification of patientswith AIN at risk of progression to invasive analcancer, and hence their subsequent manage-ment.

MethodsThirty two anal epithelial specimens werestudied. These comprised: nine normal analepithelium, five AIN I, 13 AIN III and fivesquamous cancers. These were taken from thearchival files of the Histopathology Depart-ment at the Northern General Hospital, Shef-field. The histology on all specimens wasreviewed by one consultant pathologist with an

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Ogunbiyi, Scholefield, Sharp, Ginsberg, Rogers

interest in anogenital neoplasia. The specimenshad been fixed in 10% buffered formalin-phosphate and paraffin wax embedded. Sec-tions (5 gum thick) were dewaxed in xylene andhydrated through graded ethanols to deionisedwater. Adjacent sections from each block werestained with haematoxylin and eosin and forsilver binding NOR associated protein, as

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¶ described by Smith and Crocker.'3 TheAgNOR staining solution was prepared bydissolving a 2% solution of gelatin in 1%formic acid. This solution was then mixed in aratio of 1:2 with 50% aqueous silver nitrate.

*̂ ^ The tissue sections were immersed in the finalworking solution and left for 30 minutes atroom temperature in a dark room. The slideswere then fixed, washed in distilled water,

w dehydrated in ascending grades of ethanol andmounted. The presence of NORs is indicated

* W by the appearance of black silver granule(s) in* * the nucleus (fig 1).

4v ;AgNORs present in the nuclei of 100 con-secutive normal or atypical anal epithelial cellswere counted at a magnification of x 100using an oil immersion lens by a singleobserver. Two methods of AgNOR enumera-tion were evaluated by two observers in apreliminary series of 10 specimens. In the first

* method the actual numbers of AgNORs werequantified by counting each apparent dot evenwhen they touched or overlapped. The inter-observer variation was 25-30% using thismethod. The second method involved count-ing the number of separable black dots in eachnucleus. Overlapping dots were counted asone. Using this method, interobserver varia-tion was 5-8%. The second method ofAgNORenumeration was therefore felt to be morereliable and used for the purpose of this study.The number of nuclei to be counted in eachspecimen was determined by the cumulativemeans technique. Using this procedure, it wasshown that the mean number of AgNORs

q would not have been altered in any lesion bycounting more than 100 nuclei.

All the data were analysed using the Mann-Whimey U test for non-parametric data.

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Figure 1 AgNORs in (A) normal anal epithelium and (B) AIN I. The AgNup as single or multiple black dots in the nucleus (shown by arrows).

¶ ResultsFor each specimen, the cumulative meanstechnique was used to determine the numberof nuclei that needed to be counted before themeans became stable. The mean was arbitrarilydefined as being stable when the mean reachedand stayed within ± 5% of the final mean (n =100). Figure 2 illustrates moving average plots

f of typical examples of normal anal epithelium,AIN III, and invasive squamous carcinoma ofthe anus. In the illustrated examples the meanvalues became stable after 30 nuclei had beencounted for normal anal epithelium and 50nuclei for both AIN3 and anal squamouscancer. The median numbers of nuclei countedin each group before the mean became stableare as follows: normal anal epithelium (n = 9)35 (range 20-70); AIN I (n = 5) 40 (range30-70); AIN III (n = 13) 50 (range 30-70);

'ORs show and invasive anal squamous cell carcinoma(n = 5) 50 (range 20-70).

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The scattergram in fig 3 depicts the meanAgNOR counts for each specimen in all fourdiagnostic groups. In all specimens AgNORs

t*W were clearly visible as black dots of varying4 sizes in the nuclei. These were arranged into

one or more clusters or as individual satel-lites-extranuceolar. In AIN III and invasiveanal squamous cancer specimens there weremore AgNOR clusters as well as extranucleolar

*# AgNORs present compared with normal analepithelium and AIN I. The final median valuesfor each group are shown in table 1. Althoughthere is a progressive increase in the medianAgNOR values, there is a considerable degreeof overlap between the four groups as illus-

* trated in the scattergram. Using the Mann-Whitney U test for non-parametric data (table2), there were significant differences between

1* AgNOR counts in anal cancer and normal analepithelium (p < 005), AIN III and normalanal epithelium (p < 0 005), and AIN III andAIN I (p < 005). No significant differenceswere observed between AIN I and normal analepithelium, anal cancer and AIN I, and cancerand AIN III.

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DiscussionAlthough possible parallels between AIN andCIN have been suggested,6 the natural historyofAIN is still unknown. However, AIN III hasbeen detected in resection specimens for squa-mous anal cancer.4 14 These lesions were

usually found adjacent to the tumours as wellas in areas separated from the tumour by

* normal mucosa. AIN seems to be fairly com-

mon in certain at risk groups, and in view ofthe rarity of anal carcinoma (3% of bowel

** cancers), most of the AIN lesions seem toregress or remain static. This poses problems inthe management ofAIN lesions, as treating alllesions would result in unnecessary treatmentof large numbers of patients. On the otherhand, the possibility that a small number ofpatients with untreated AIN may progress toinvasive cancer has to be considered. How,then, do we determine which AIN lesions are

likely to progress to invasive cancer? Ideally amarker identifying abnormal anal cells wouldaid in the diagnosis and follow up of patientswith MIN.

*t ti*e This study gives the first description ofAgNOR counts in AIN and invasive analsquamous cancer. We have tried to evaluatethe usefulness of AgNORs in differentiatingbetween anal neoplastic lesions and hence thepossibility of using the method as a marker infollowing the progress of AN lesions. TheAgNOR technique has been used by cytoge-neticists for over a decade, although it has onlyrecently been applied in histopathology." The

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Figure 1 continued AgNORs in (C) AIN III and (D) invasive anal squamouscarcinoma. The AgNORs show up as single or multiple black dots in the nucleus (shownby arrows). The dots in AIN III and anal cancer are smaller andform numerous clusterscompared with normal anal epithelium andAIN I.

Table 1 Median AgNOR values

Median CI

Normal 2-15 95% (1-89-3-94)AIN I 3-21 95% (2-89-7-14)AIN III 4-32 95% (4-00-8- 10)Carcinoma 5-51 95% (2-48-10-62)

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Ogunbiyi, Scholefield, Sharp, Ginsberg, Rogers

6 Anal Ploton et al (1986) were the first to suggest thatcarcinoma interphase AgNOR counts could indicate

5 malignant disease when they showed that~i_ AgNOR counts in prostatic carcinoma greatly

AIIITNIIIexceeded that in benign prostatic hyperpla-

4 AINia. 1s\_+ Since then there have been several studies

3/ describing the application of silver staining

methods for the demonstration of AgNORs inNormal

histological material from a wide range of2 Normal diseases. These studies have shown that malig-

nant cells can be distinguished from corre-sponding benign or normal cells on the basis of

1 a higher quantity of interphase AgNORs. 9 20Our results have shown that although there

o2,0, , , is a progressive increase in the median numbero 10 20 30 40 50 60 70 so 90 100 of AgNOR counts in normal, AIN I, AIN III

Nuclei counted and invasive cancer, there is a high degree ofoverlap between the groups, particularly

re 2 Cumulative means technique: moving average plots of typical examples of between AIN III and invasive cancer. Fornal anal epithelium, AIN III and invasive anal squamous cell carcinoma. Thecal bars indicate the number of nuclei counted to achieve a stable mean. practical purposes, therefore, AgNOR counts

are of limited value in discriminating betweengrades of AIN and invasive cancer and areunlikely to be suitable as a marker of disease

14 ~ progression. These results are similar to those13 - x obtained by Darne et al,21 who evaluated2 - AgNORs in normal endocervix, adenocarci-11 - x noma in situ (AIS), and invasive adenocarci-0 x noma of the cervix. They found that although9 X

xAgNOR counts differentiated between normal

8 - x endocervical cells on the one hand and AIS7 x x and invasive adenocarcinoma on the other,6 X there was a significant degree of overlapXx x between cases of AIS and invasive adenocarci-4 4 X noma. This suggested that although AIS was a3X

x5t potential premalignant precursor of invasive

x adenocarcinoma, the assessment of AgNORs2- was of limited use in discriminating between1 - the histological types of cervical carcinoma.O The evaluation of AgNORs in CIN has also

Normal AIN AIN III Anal 2carcinoma been shown to be of limited diagnostic value.'2In that study the mean number of AgNORs

tre 3 Scattergram to show the distribution of total numbers ofAgNORs per 100 cells was shown to increase steadily in the threezch case examined. The horizontal bars indicate the median for each group. grades of CIN with some significant differ-

ences between the groups. However, there wasoverlap between the grades, thereby making

NORs are present on the short arms of five this method of limited dignostic value.chromosomes 13, 14, 15, 21 and 22 in man, Recently, studies of oncogene expression inand have allowed various genetic defects in cervical intraepithelial neoplasia (CIN) andmetaphase chromosome spreads to be ana- invasive cancer of the cervix have shownlysed. The nature of the silver staining NOR- molecular alterations of c-myc oncogene inassociated proteins shown by the AgNOR carcinoma of the cervix as well as over-technique is not fully known, although it is expression of its protein product p62.22 23thought they may be related to proteins such as Crook et al24 have also demonstrated similarRNA polymerase I,15 C23 (nucleolin), and changes in anal squamous cell carcinoma,B23 protein.'667 The exact function of these although no changes were observed in the fiveproteins is uncertain, although it is thought specimens ofAIN III examined. C-myc onco-that they may have some regulatory function in gene expression may serve as a marker ofcontrolling the transcription of the genes for disease progression, and studies into this areribosomal RNA and hence protein synthesis. currently being undertaken in this unit.

Table 2 Statistical analysis ofAgNOR counts using Mann-Whitney U test

Point estimate CI P valueNormal v AlN I -1 10 95 (- 2-941, 0-931) >0 05Normal v AIN III - 2-270 95 (- 5-701, -1-350) < 0-005Normal v anal carcinoma -3-130 95 (- 6-419, -0-329) < 0-05AIN I v AIN III -1 110 95 (- 5399, -0-220) < 0 05AIN I v anal carcinoma -2-110 95 (- 7-410, 1-632) > 0-05AIN III v anal carcinoma -0-600 95 (- 2-098, 3-679) > 0-05

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4 Palefsky JM, Gonzales J, Greenblatt RM, Ahn DK, Hol-lander H. Anal intraepithelial neoplasia and anal papillo-mavirus infection among homosexual males with GroupIV HIV disease. JAMA 1990;263:291 1-16.

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