Path. Res. Pract. 186, 254-259 (1990)
Nucleolar Organiser Regions in Endometrial Hyperplasia and Neoplasia A. Coumbe, B. P. Mills and C. L. Brown Department of Morbid Anatomy, The London Hospital, Whitechapel, U.K.
SUMMARY
Nucleolar organiser regions (NORs) were counted in the nuclei of both epithelial and stromal cells in cases ofendometrial hyperplasia and carcinoma. The scores for each case were expressed as a ratio. No significant difference was found between the ratios for cystic hyperplasia and hyperplasia with architectural atypia, confirming the view that these are morphological variants ofthe same benign hyperplastic process. The ratios for hyperplasia with cytological atypia and adenocarcinoma differed significantly from the benign hyperplasias. This adds support to the view that hyperplasia with cytological atypia should be considered a neoplastic condition.
Introduction Histological diagnosis of mild endometrial hyperplasia and poorly differentiated adenocarcinoma is usually straightforward. However, atypical hyperplasia, carcinoma-in-situ and well differentiated adenocarcinoma comprise a group of borderline conditions that can be difficult to distinguish and accurately to place4. Furthermore a confusing array of classifications has been used by different pathologists and no absolute diagnostic criteria are recognised1, 2, 12, 13, 16, 18. In a review of this difficult area Fox and Buckley9 attempted to rationalise the nomenclature. They recognised four types of endometrial hyperplasia: 1. Cystic glandular hyperplasia 2. Adenomatoid hyperplasia 3. Glandular hyperplasia with architectural atypia 4. Glandular hyperplasia with cytological atypia and argued that the last of these should be grouped with in-situ adenocarcinoma as intra-endometrial carcinoma. There are important prognostic and therapeutic implications attached to the diagnosis of these borderline lesions and any technique which facilitates their recognition will clearly be welcome. Nucleolar organiser regions are loops of DNA which have been shown, by in-situ hybridisation, to transcribe to ribosomal RNNo. In man, five acrocentric chromosomes 0344-0338/90/0186-0254$3.50/0
(13,14,15,21 and 22) bear NORs. A simple argyrophilic technique to demonstrate NORs was introduced in 1975 11 and has been widely used by cytogeneticists in the investigation of chromosome disorders, especially trisomies. This technique was further modified by Ploton et a1. 15 making it suitable for use on resin and paraffin wax embedded tissue. The argyrophil method for NOR staining (AgNOR technique) identifies NOR-associated proteins rather than NORs themselves. The silver binding is highly specific and occurs by means of sulphhydryl groups. The proteins associated with NORs include RNA polymerase I, which catalyses the transcription of ribosomal DNA to RNA20. Unusual NOR patterns were observed in cytogenetic studies of certain malignancies and a small study suggested that in prostatic carcinoma several AgNOR nuclear dots were present whereas in hyperplastic prostate sections only two dots were seen15. The observation that an increase in NOR numbers occurs in hyperplastic and neoplastic lesions together with the significance of NORs as markers of ribosomal DNA transcription has led to the belief that their numbers reflect cellular proliferative activity. NOR numbers may also be expected to be increased in aneuploid tumours. We were attracted to the idea of looking at NORs in endometrial hyperplasia by a report that ribosomal genes, ©
1990 by Gustav Fischer Verlag, Stuttgart
Nucleolar Organiser Regions in Endometrial Hyperplasia and Neoplasia· 255
and hence NORs, may be hormonally regulated? In cystic hyperplasia excessive oestrogenic stimulation causes increased proliferative activity in both glandular epithelium and stroma, with increased NORs in both. Conversely at the other end of the spectrum the neoplastic proliferation of epithelium in adenocarcinoma could be expected to greatly exceed that of the stroma. By counting NORs in the nuclei of both epithelium and stroma and expressing these as a ratio we would derive a proliferation index for epithelium relative to its surrounding stroma. Our aim in this study was to determine whether an NOR ratio of this sort would be more discriminating than an epithelial NOR score in the distinction between benign and malignant endometrial proliferations. We also hoped to further define the position of the atypical hyperplasias within the spectrum of endometrial proliferative lesions. Material and Methods The cases examined in this study were drawn from the files at the London Hospital. The reports of this archive material are coded by the SNOP system and since 1980 have been stored on computer. Cases from 1980 onwards to which the diagnoses of cystic, unspecified or atypical hyperplasia, adenocarcinoma insitu or adenocarcinoma had been attached were examined. Each was reviewed and assigned to one of the categories described by Fox and Buckley 9. Cases of simple (cystic glandular) hyperplasia (24) were readily recognised (Fig. 1). However, four cases to which this label had
Fig. 1. Cystic Glandular Hyperplasia. a: H&E,x 100jb: AgNOR, x 1000.
been attached were noted to contain thick-walled vessels and rather inactive stroma. These were considered to be fragments of endometrial polyps showing cystic change. Architectural atypia (8 cases) was diagnosed when large crowded glands with prominent outpouching but a regular epithelial lining were seen (Fig. 2). The features used to recognise cytological atypia (6 cases) were crowded "back to back" glands with true multilayering, intraluminal budding and atypical cells with rounded nuclei and prominent nucleoli (Fig. 3). No attempt was made to define a sub-category of adenocarcinoma in-situ because myometrial invasion could not be properly assessed in curettage material. Adenocarcinoma (18 cases) was identified when true intraglandular bridges (without stromal support), sheets of atypical cells within glands and polymorphs within gland lumens were seen (Fig. 4). Sections were cut at 3 Il, dewaxed and stained for 25-30 min at 20°C using the single step argyrophil technique. Background silver deposits were reduced by staining the sections back to back in a covered Coplin jar. Initially a neutral red counterstain was used but counting was found to be easier without. For each case the slides were examined under oil immersion and NORs were counted in 100 nuclei of each cell type. The total number of individually discernible dots were counted, clusters were scored as the total of their constituent resolvable dots.
Results The mean NOR scores and ratios for each category are given in Table 1, together with standard deviations. The
256 . A. Coumbe, B. P. Mills and C. L. Brown
Fig. 2. Architectural Atypia. a: H & E, x 100; b: AgNOR, X 1000.
Fig. 3. Cytological Atypia. a: H & E, x 400; b: AgNOR, x 1000.
Nucleolar Organiser Regions in Endometrial Hyperplasia and Neoplasia . 257
Fig. 4. Adenocarcinoma. a: H & E, X 400; b: AgNOR, X 1000.
results are presented diagrammatically in Figs. 5 and 6. The mean value for each category is shown as a narrow black line and the hatched areas within the horizontal bars indicate two standard deviations to either side of the mean giving the 95% confidence limits. Analysis of variance of these data is complicated by the fact that more than two groups of measurements are presented. Student's t test is therefore not applicable and instead Scheff/?s F test was performed. For the NOR ratios this showed no significant difference, at the 5% level, between cystic hyperplasia or hyperplasia with architectural atypia. However, significant differences were present between these and hyperplasia with cytological atypia and adenocarcinoma.
Table 1. Mean NOR scores for glandular epithelium in each category together with the standard deviation. Also given are the ratio of glandular epithelial NOR score to stromal score for each type Number Glandular epithelium Cystic hyperplasia Architectural atypia Polyps Cytological atypia Adenocarcinoma
CYs tt c Hyp erpI asi a,...,...,r-r>"T7-rr"""""'77"77""TTr-r>'777'1
Cystic Hyperplasia
Architect ural At Ypia r-r>......,..-rr"""""'77"77""TTr-r>"7"?I
Architectural Atypi a
Cytological Atypia ~ ~
Cytological Atypia
Adenocarcinoma ~~
Adenocarci noma
V/JJJJflJ///O/~A
3
4
5
6
3.77 3.73 3.59 5.20 4.65
± ± ± ± ±
1.33 1.24 0.51 0.94 0.80
1.19 1.15 1.83 2.45 2.79
± 0.18 ± 0.18 ± 0.17 ± 0.36 ± 0.29
V/fl/A'/fl/A
V////////J/////ff~~~
2
24 8 4 6 18
Gland/stroma ratio
V/(/Uf /(/(/
Nucleolar Organiser Regions in Endometrial Hyperplasia and Neoplasia A. Coumbe, B. P. Mills and C. L. Brown Department of Morbid Anatomy, The London Hospital, Whitechapel, U.K.
SUMMARY
Nucleolar organiser regions (NORs) were counted in the nuclei of both epithelial and stromal cells in cases ofendometrial hyperplasia and carcinoma. The scores for each case were expressed as a ratio. No significant difference was found between the ratios for cystic hyperplasia and hyperplasia with architectural atypia, confirming the view that these are morphological variants ofthe same benign hyperplastic process. The ratios for hyperplasia with cytological atypia and adenocarcinoma differed significantly from the benign hyperplasias. This adds support to the view that hyperplasia with cytological atypia should be considered a neoplastic condition.
Introduction Histological diagnosis of mild endometrial hyperplasia and poorly differentiated adenocarcinoma is usually straightforward. However, atypical hyperplasia, carcinoma-in-situ and well differentiated adenocarcinoma comprise a group of borderline conditions that can be difficult to distinguish and accurately to place4. Furthermore a confusing array of classifications has been used by different pathologists and no absolute diagnostic criteria are recognised1, 2, 12, 13, 16, 18. In a review of this difficult area Fox and Buckley9 attempted to rationalise the nomenclature. They recognised four types of endometrial hyperplasia: 1. Cystic glandular hyperplasia 2. Adenomatoid hyperplasia 3. Glandular hyperplasia with architectural atypia 4. Glandular hyperplasia with cytological atypia and argued that the last of these should be grouped with in-situ adenocarcinoma as intra-endometrial carcinoma. There are important prognostic and therapeutic implications attached to the diagnosis of these borderline lesions and any technique which facilitates their recognition will clearly be welcome. Nucleolar organiser regions are loops of DNA which have been shown, by in-situ hybridisation, to transcribe to ribosomal RNNo. In man, five acrocentric chromosomes 0344-0338/90/0186-0254$3.50/0
(13,14,15,21 and 22) bear NORs. A simple argyrophilic technique to demonstrate NORs was introduced in 1975 11 and has been widely used by cytogeneticists in the investigation of chromosome disorders, especially trisomies. This technique was further modified by Ploton et a1. 15 making it suitable for use on resin and paraffin wax embedded tissue. The argyrophil method for NOR staining (AgNOR technique) identifies NOR-associated proteins rather than NORs themselves. The silver binding is highly specific and occurs by means of sulphhydryl groups. The proteins associated with NORs include RNA polymerase I, which catalyses the transcription of ribosomal DNA to RNA20. Unusual NOR patterns were observed in cytogenetic studies of certain malignancies and a small study suggested that in prostatic carcinoma several AgNOR nuclear dots were present whereas in hyperplastic prostate sections only two dots were seen15. The observation that an increase in NOR numbers occurs in hyperplastic and neoplastic lesions together with the significance of NORs as markers of ribosomal DNA transcription has led to the belief that their numbers reflect cellular proliferative activity. NOR numbers may also be expected to be increased in aneuploid tumours. We were attracted to the idea of looking at NORs in endometrial hyperplasia by a report that ribosomal genes, ©
1990 by Gustav Fischer Verlag, Stuttgart
Nucleolar Organiser Regions in Endometrial Hyperplasia and Neoplasia· 255
and hence NORs, may be hormonally regulated? In cystic hyperplasia excessive oestrogenic stimulation causes increased proliferative activity in both glandular epithelium and stroma, with increased NORs in both. Conversely at the other end of the spectrum the neoplastic proliferation of epithelium in adenocarcinoma could be expected to greatly exceed that of the stroma. By counting NORs in the nuclei of both epithelium and stroma and expressing these as a ratio we would derive a proliferation index for epithelium relative to its surrounding stroma. Our aim in this study was to determine whether an NOR ratio of this sort would be more discriminating than an epithelial NOR score in the distinction between benign and malignant endometrial proliferations. We also hoped to further define the position of the atypical hyperplasias within the spectrum of endometrial proliferative lesions. Material and Methods The cases examined in this study were drawn from the files at the London Hospital. The reports of this archive material are coded by the SNOP system and since 1980 have been stored on computer. Cases from 1980 onwards to which the diagnoses of cystic, unspecified or atypical hyperplasia, adenocarcinoma insitu or adenocarcinoma had been attached were examined. Each was reviewed and assigned to one of the categories described by Fox and Buckley 9. Cases of simple (cystic glandular) hyperplasia (24) were readily recognised (Fig. 1). However, four cases to which this label had
Fig. 1. Cystic Glandular Hyperplasia. a: H&E,x 100jb: AgNOR, x 1000.
been attached were noted to contain thick-walled vessels and rather inactive stroma. These were considered to be fragments of endometrial polyps showing cystic change. Architectural atypia (8 cases) was diagnosed when large crowded glands with prominent outpouching but a regular epithelial lining were seen (Fig. 2). The features used to recognise cytological atypia (6 cases) were crowded "back to back" glands with true multilayering, intraluminal budding and atypical cells with rounded nuclei and prominent nucleoli (Fig. 3). No attempt was made to define a sub-category of adenocarcinoma in-situ because myometrial invasion could not be properly assessed in curettage material. Adenocarcinoma (18 cases) was identified when true intraglandular bridges (without stromal support), sheets of atypical cells within glands and polymorphs within gland lumens were seen (Fig. 4). Sections were cut at 3 Il, dewaxed and stained for 25-30 min at 20°C using the single step argyrophil technique. Background silver deposits were reduced by staining the sections back to back in a covered Coplin jar. Initially a neutral red counterstain was used but counting was found to be easier without. For each case the slides were examined under oil immersion and NORs were counted in 100 nuclei of each cell type. The total number of individually discernible dots were counted, clusters were scored as the total of their constituent resolvable dots.
Results The mean NOR scores and ratios for each category are given in Table 1, together with standard deviations. The
256 . A. Coumbe, B. P. Mills and C. L. Brown
Fig. 2. Architectural Atypia. a: H & E, x 100; b: AgNOR, X 1000.
Fig. 3. Cytological Atypia. a: H & E, x 400; b: AgNOR, x 1000.
Nucleolar Organiser Regions in Endometrial Hyperplasia and Neoplasia . 257
Fig. 4. Adenocarcinoma. a: H & E, X 400; b: AgNOR, X 1000.
results are presented diagrammatically in Figs. 5 and 6. The mean value for each category is shown as a narrow black line and the hatched areas within the horizontal bars indicate two standard deviations to either side of the mean giving the 95% confidence limits. Analysis of variance of these data is complicated by the fact that more than two groups of measurements are presented. Student's t test is therefore not applicable and instead Scheff/?s F test was performed. For the NOR ratios this showed no significant difference, at the 5% level, between cystic hyperplasia or hyperplasia with architectural atypia. However, significant differences were present between these and hyperplasia with cytological atypia and adenocarcinoma.
Table 1. Mean NOR scores for glandular epithelium in each category together with the standard deviation. Also given are the ratio of glandular epithelial NOR score to stromal score for each type Number Glandular epithelium Cystic hyperplasia Architectural atypia Polyps Cytological atypia Adenocarcinoma
CYs tt c Hyp erpI asi a,...,...,r-r>"T7-rr"""""'77"77""TTr-r>'777'1
Cystic Hyperplasia
Architect ural At Ypia r-r>......,..-rr"""""'77"77""TTr-r>"7"?I
Architectural Atypi a
Cytological Atypia ~ ~
Cytological Atypia
Adenocarcinoma ~~
Adenocarci noma
V/JJJJflJ///O/~A
3
4
5
6
3.77 3.73 3.59 5.20 4.65
± ± ± ± ±
1.33 1.24 0.51 0.94 0.80
1.19 1.15 1.83 2.45 2.79
± 0.18 ± 0.18 ± 0.17 ± 0.36 ± 0.29
V/fl/A'/fl/A
V////////J/////ff~~~
2
24 8 4 6 18
Gland/stroma ratio
V/(/Uf /(/(/
