Path. Res. Pract. 191,381-390 (1995)
Nucleolar and AgNOR-analysis of Prostatic Intraepithelial Neoplasia (PIN), Atypical Adenomatous Hyperplasia (AAH) and Prostatic Carcinoma ~IB. Helpap and C. Riede Institute of Pathology, General Hospital of Singen, Academic Instructional Hospital of the University of Freiburg, Federal Republic of Germany
SUMMARY Atypical glandular proliferations of the prostate are found in combination with prostate carcinomas but also with benign prostatic hyperplasia (BPH). These atypical glandular lesions that mimic glandular carcinoma within the anterocentral part of the prostate (transition zone) are named "atypical adenomatous hyperplasia (AAH)." The term for atypical ductal and acinar mainly papillary and/or cribriform proliferations in the peripheral zone is "prostatic intraepithelial neoplasia (PIN)." In this study the significance of the status of nucleoli and the number of silver-stained nucleolar organizing regions (AgNOR) as a method for measuring proliferative activity was investigated in biopsies of AAH, PIN and carcinomas of different grades of malignancy. The aim was to prove whether there are relationships of AAH to low grade carcinomas on the one hand and of PIN to high grade carcinomas on the other hand. The frequency of nuclei with small nucleoli was low in BPH (0.6%). The values increased to 8.3% in AAH, and 28.7% in PIN. The frequency of nuclei with prominent nucleoli in low grade carcinomas was 41.7% and in high grade carcinomas 81.1 %. Usually, one single nucleolus was found per nucleus. Multiple prominent nucleoli were found in high grade carcinomas and in some cases of high grade PIN only. The result of AgNOR-analysis was similar: low numbers in BPH, low to moderate values in AAH, moderate values in low grade carcinomas and high values in PIN, as well as high grade carcinomas. Besides correlation between topography, histology and cytology, the similarities of nucleolar and AgNOR-analysis between PIN and high grade carcinomas support the idea that PIN is the precursor of the peripheral prostatic carcinoma. The histological, cytological and cell kinetical features of AAH are intermediate between BPH and low grade carcinoma of the prostate. Therefore, the findings to date are inconclusive and further follow up studies are necessary to prove if AAH may be a precursor of transition zone carcinoma of the prostate.
* Paper presented at first international consultation meeting on prostatic intra epithelial neoplasia and the origins of prostatic carcinoma, Ancona, Italy, Sept. 11-12, 1994. Meeting of the International Society of Urologic Pathology Prognostic Factors in Carcinoma of the Prostate. Hong Kong Oct. 10-14. 1994. © 1995 by Gustav Fischer Verlag, Stuttgart
Introduction Atypical glandular and ductal proliferations of the prostate may be combined with carcinomas but also with benign prostatic hyperplasia (BPH) without carcinomas. The terms and significances of these atypical 0344-0338/95/0191-0381 $3.50/0
382 . B. Helpap and C. Riede
lesions which resemble low and high grade carcinoma are in discussion2, 8,9, 10, 12, 13,23,44,64. For the atypical lesion in the anterocentral and/or transition zone of the prostate, the term "atypical adenomatous hyperplasia (AAH)" is now in use, and in the dorsoperipheral zone of the prostate, "prostatic intraepithelial neoplasia (PIN)." The AAH is characterized by newly formed microglandular structures with unilayered basal cells and secretory cells with different grades of cellular, nuclear and nucleolar atypias. The histological and cytological pattern is often very similar to microglandular carcinoma, especially of low malignancy9,23. The prostatic intra epithelial neoplasia is characterized by cellular proliferations within preexisting glands and ducts with mild, moderate or marked cellular, nuclear, as well as nucleolar atypias. Furthermore, PIN is characterized by epithelial crowding and stratification with irregular spacing, enlarged nuclei and prominent nucleoli. In high grade PIN, crowding and stratification is more pronounced. The architectural pattern is tufting, micro papillary, cribriform or flat. The cytological pattern is similar to carcinoma. Basal cell layers may show some disruption 6,7,8. The preneoplastic character of both of these lesions has been studied by various experimental methods like autoradiography, DNA-cytometry and immunohistochemistry3,4,17,21,26,45,47,50,51,54,60. But there do not exist findings about a rank order of AAH and/or PIN to low and high grade carcinomas. Such relationships may be helpful for the assessment of prognostic values in diagnosis and therapy for the urologists. Nucleolar analyses and the use of NOR silver staining as a parameter of cell proliferation are reliable methods to estimate the grade of malignancy and the cell proliferation respectivel y14, 15,18,22,24,25,29,36,38,39,40,43,46,53,60,62. In this study, therefore, the significance of the status of nucleoli and the number of silver-stained nucleolar organizer regions (AgNOR) were investigated in biopsies of AAH, PIN and carcinomas of different grades of malignancy using a WHO modified grading systeml7,31.
Subgrading We subgraded the carcinoma groups I, II and III using the modified grading system of the German pathological and urological working group "prostate cancer"31,32. The WHOgrading system was modified by introducing subgroups a and b, especially by nucleolar subgrading. The histology of usual prostatic carcinomas with glandular, cribriform and solid/trabecular pattern was scored between 0 to 3. The grade of cytological atypias like variations of nuclear size, chromatin density, nucleus!cytoplasma relation shifts and mitotic frequency, as well as the number of nuclei with prominent nucleoli and central or eccentric location of nucleoli within the nuclei, were scored between 0 to 2. Grade I well differentiated glandular carcinoma comprised subgroup Ia with minimal nuclear atypias and no prominent nucleoli, and subgroup Ib with moderate nuclear atypias and slightly increased prominence of nucleoli. Grade II carcinomas were subdivided in subgroup IIa with moderate nuclear atypia and subgroup lIb with glandular or cribriform pattern, moderate to severe nuclear atypia, and increased numbers of nucleoli mainly in eccentric positions. Grade IlIa solid/trabecular carcinoma showed severe nuclear atypias. Subgroup I1Ib corresponds to the completely undifferentiated carcinoma 29,31.
Nucleolar Analysis The number of nuclei containing nucleoli and the number of nucleoli inside each nucleus were determined after hematoxylin-eosin staining. The nucleolar size was set in relation to the nuclear size, i.e. the ratio of nucleolar and nuclear diameter was calculated. Two groups (cut off point 0.125) were distinguished. Furthermore, we evaluated the intranuclear location of the nucleoli i.e. central or eccentric/marginated position. This method of grouping is in accordance to the general practice applied by various investigators29,32,36,39,46.
AgNOR-analysis For AgNOR staining aqueous silver nitrate solution (500 gil) and gelatine (20 gil in 10 gil aqueous formic acid) were mixed 2: 1 (v/v). After staining, the slides were incubated at 37 C for 30 minutes 55 . Staining was considered satisfactory when silver stained dots (AgNORs) were clearly visible within the nucleoli 57.
AgNOR Counting Material and Methods The histological analysis of the prostatic tissue was done on 1,651 specimens obtained by punch biopsy and transurethral resection. The material was embedded in paraffin and stained with hematoxylin and eosin. We studied the frequency of BPH with and without inflammation, prostatitis, PIN and AAH with and without carcinomas, and prostatic carcinomas grade I-III with subgroups a and b. AAH and PIN were histologically classified and PIN was graded in low and high grades of atypia, according to the criteria given by Bostwick and Brawer6,7, 8, 12. Furthermore, we divided AAH into low and high grades of atypia in consideration of different grades of architectural, cellular and nuclear atypias 9, 10, 13,23,34,35.
The AgNORs were counted following the recommendation of Crocker 16 ; i.e. all AgNORs (black dots) both inside and outside nucleoli were enumerated as separate entities and included into the count. AgNOR counting was performed after focusing on the nuclear membrane and fine granular matrix. Overlapping dots were not counted. 200 cells were evaluated per slide. The AgNOR and nucleolar analyses were done at a 1000 fold magnification using an oil immersion lens and an eyepiece graticule (Leitz/Orthoplan). The results (mean values and standard deviation) were statistically validated by applying the Wilcoxon test with Holm's correction.
Results In a total of 1,651 specimens (TUR and biopsies), BPH with and without inflammation was found in
Nucleolar and AgNOR Analysis of the Prostate . 383
44.1 % and prostatitis in 5%. The values of PIN and AAH were 16.7% and 7.3%, respectively. The percentage of carcinomas was 26.9 (Tab. 1). We studied 398 cases of PIN and AAH (Figs. 1,4). 70.8% of PIN were associated with carcinomas, whereas in only 18.2% was AAH combined with carcinomas. We found high grade PIN mainly combined with high grade carcinomas in 85.5% and AAH mainly associated with low grade carcinoma in 81.8%. In cases without carcinomas low grade PIN and mild form of AAH predominated with 85.6% and 92.9%, respectively (Tab. 2, 3). The carcinomas were classified by histological and cytological pattern as well as by the grade of malignancy. Well differentiated glandular carcinomas were very rare. In most cases the glandular carcinomas were moderately differentiated with mild to moderate nuclear and nucleolar atypias. The frequency of low grade carcinomas grade Ia-lla was 35.6%. The group of high grade carcinoma Gllb-GIIIb with cribriform, solid/trabecular and undifferentiated pattern came to 64.4% (Tab. 4).
Nucleoli The frequency of nuclei with nucleoli in BPH was very low (0.6 ± 0.6%). The nucleoli were solitary, very small (ratio of nucleolar and nuclear diameter < 0.125) and in central position of the nucleus. The frequency increased up to 1.4% ± 0.4% and 8.3% ± 1.6% in low and high grade AAH respectively. The nucleoli were again small « 0.125) and in central position (Fig. 2). The values in low and high grade PIN were 4.6% ± 2.4% and 28.7% ± 6.4% respectively. The nucleoli were mostly singular. The differences between AAH and PIN were significant (p <0.001). Some cases of high grade PIN had multiple and prominent nucleoli (> 0.125) in marginated or eccentric position (Fig. 3,4). The frequency of nuclei with nucleoli in GIa-carcinomas was 9.5% ± 3.6%; in GIb 17.8% ± 4.8%, and in GIla 38.4% ± 3.8%. The values of GlIb, GllIa-carcinomas and GIIIb-carcinomas were 60.6% ± 6.2%, 87.4% ± 7.8%, and 89.2% ± 6.8%, respectively. The number of nucleoli was low in GI-GIIa-carcinomas, but high in GlIb and GIll-carcinomas. The nucleoli were prominent (> 0.125), sometimes multiple and in central and/or eccentric position (Fig. 5, 6). The difference between GIla and GlIb-carcinomas was highly significant (p <0.001) (Tab. 5).
AgNORs The results for AgNOR paralleled those of nucleoli. The lowest frequency could be observed in BPH (2.6 ± 0.5). We found no significant differences of AgNOR number between basal and luminal cells. There was a significant increase of the number of AgNORs per nucleus in AAH (3.7 ± 1.1) and PIN (6.8 ± 0.6) (p < 0.001). For all subgraded carcinomas mean values of AgNORs were significantly different from BPH: GIa 5.0 ± 0.5, GIb 4.6 ± 0.9, GIla 5.4 ± 0.8, GlIb 8.3 ± 0.5, GllIa 9.4 ± 0.9. The difference between GIla and
Table 1. Histological Diagnoses of Prostatic Tissue Specimens (n = 1,651) Diagnosis
Benign prostatic hyperplasia (BPH) without inflammation with inflammation
Prostatitis Prostatic intra epithelial neoplasia (PIN) without carcinoma with carcinoma
Atypical adenomatous hyperplasia (AAH) without carcinoma with carcinoma
44.1 5.0 16.7
Table 2. Low and high grade prostatic intra epithelial neoplasia (PIN) with and without carcinoma (n = 277) Grading of PIN
Without carcinoma n = 81
With carcinoma * n = 196
Low grade High grade
* Mainly high grade of malignancy (GIIb-GIll).
Table 3. Atypical adenomatous hyperplasia with and without carcinoma (n = 121) Grading of AAH
Without carcinoma n = 99
With carcinoma * n = 22
Low grade High grade
* Mainly low grade of malignancy (GIa-GIIa).
Table 4. Classification and grading of prostatic carcinoma (n = 445) Carcinoma type
Well differentiated, glandular carcinoma Moderately differentiated, glandular carcmoma
Poorly differentiated, glandular carcinorna with moderate nuclear and nucleolar atypia
Poorly differentiated, glandular or cribriform carcinoma with marked nuclear and nucleolar atypia Solid-trabecular carcinoma with marked atypia
384 . B. Helpap and C. Riede
Fig. 1. Nodule of atypical adenomatous hyperplasia (AAH) with architectural disturbance and normal glands of BPH. Hematoxylin-eosin x 130. - Fig. 2. Atypical adenomatous hyperplasia (AAH) with variable size of gland back to back position, conspicuous basal cell layer, variation of nuclear size and few small nucleoli. (Hematoxylin-eosin x 400). Table 5. Nucleolar pattern of BPH, AAH, PIN, and carcinomas Histology
% of nuclei with nucleoli
Location in nucleus
Typical hyperplasia (BPH)
0.6 ± 0.6
Atypical adenoma to us Hyperplasia (AAH) Low grade High grade
1.4 ± 0.4 8.3 ± 1.6
4.6 ± 2.4 28.7 ± 6.4
2 65 91
9.5 ± 3.6 17.8 ± 4.8 38.4 ± 3.8 60.6 ± 6.2 87.4 ± 7.8 89.2 ± 6.8
solitary solitary solitary/multiple multiple multiple multiple
central central centraVeccentric eccentric eccentric eccentric
Prostatic intraepithelial neoplasia (PIN) Low grade High grade Carcinomas Grade Ia Grade Ib Grade lIa Grade lIb Grade lIla Grade IIIb
Nucleolar and AgNOR Analysis of the Prostate . 385 I
, ... \
Fig. 3. Prostatic intraepithelial neoplasia (PIN) with cribriform pattern and distinct nuclear atypias with eccentric nucleoli. Hematoxylin-eosin x 130. - Fig. 4. Prostatic intraepithelial neoplasia (high grade PIN) with epithelial cell crowding and stratification, irregular spacing and cribriform pattern. Fragmented basal cell layer, prominent nucleoli in central and eccentric position. Few apoptotic bodies. Hematoxylin-eosin x 400.
Table 6. AgNOR pattern of prostatic tissue Histology
AgNOR per nucleus
Typical hyperplasia (BPH) Atypical adenomatous hyperplasia (AAH) moderate-marked Prostatic intraepithelial neoplasia (PIN) moderate-marked
2.6 ± 0.5 3.7 ± 1.1
6.8 ± 0.6
Carcinomas Grade Ia-IIa
5.0 ± 0.8
8.9 ± 0.7
Glib-carcinomas was highly significant (p < 0.001) (Fig. 7, 8, 9). The differences between GIa, GIb and GIla-carcinomas were not significant, nor were those
between Glib and GIll-carcinomas. Therefore the values of Gla, Glb and GIla-carcinomas as well as GIIband GIll-carcinomas, were pooled and the carcinomas divided into two groups. The values of the group of Gla, Glb and GIla-carcinomas were significantly different to the group of Glib and GIll-carcinomas (p < 0.001). There were no significant differences between AAH and Gla, Glb and GIla-carcinomas, and PIN versus Glib and GIll-carcinomas. AAH and PIN were, however, significantly different (Tab. 6). Discussion In this study we demonstrated significant differences in nucleolar and AgNOR frequencies between two groups of carcinomas of the prostate with low and high grade of malignancy. Prostatic carcinomas of low malignancy (GIa, b and GIla) had only few nuclei with a
386 . B. HeJpap and C. Riede
single nucleolus in central position and were characterized by a small number of AgNORs, while highly malignant prostatic carcinomas (GlIb-III) had a high frequency of nuclei predominantly with more than one nucleolus in mainly eccentric position and a large number of AgNORs per nucleus. These findings correlate with compilated values of the literature. The range of AgNOR-number per nucleus in BPH is 1.6-2.5, in AAH 1.7-5.6, in PIN (high grade) 3.2-7.4, in low grade carcinomas 2.6-7.9 and in high grade carcinomas 3.1-11.4. The mean values of nucleolar frequency, size of nucleoli and the number of nucleoli within the nuclei, according to the compilated literature, confirm these relationships with increase of malignancy. There is an increase of frequency of nuclei with nucleoli and an increase in size and numbers of nucleoli. Furthermore, the shifting of nucleoli could be observed from central to eccentric position within the nuclei in a similar way, as in our own cases 46 • This division of prostatic carcinomas in two groups with low and high malignancy has a prognostic signif-
icance. Kobayashi and coworkers 38 have divided patients with prostatic carcinoma and low or high value of AgNOR in two groups. Patients with carcinomas having low AgNOR values had significantly longer stable durations of cancer follow up than the group of patients with carcinomas with high AgNOR values. Our AgNOR analysis, as well as previous cell kinetic studies and DNA cytometric findings, and the nucleolar subgrading support the idea that patients with cancer of the prostate have to be divided in two groups, one with low and the other with high risk. The 10 year survival rate in the literature of the low risk group is 6070% and of the high risk group 20-30%37. In one of our own studies on 226 patients, the 10 year death rates due to prostate carcinoma (DoD) were 2.8% for the low risk cancer group (GI-GlIa) and 38,2% for the high risk cancer group (GlIb-GIll) 33. The different rate of response and survival rate after hormonal or radiation treatment supports the division into low and high risk groups by cell kinetical studies with Ki67 proliferation antigen20,28, 52, 58.
Fig. 5. Glandular prostatic carcinoma with few nucleoli in central and eccentric position within the nuclei. Grade IIa. Hematoxylin-eosin x 625. - Fig. 6. Glandular prostatic carcinoma with high number of nucleoli in central and eccentric position within the nuclei. Grade GlIb. (Hematoxylin-eosin x 625).
Nucleolar and AgNOR Analysis of the Prostate' 387
Fig. 7. Glandular prostatic carcinoma with few AgNORs within the nuclei. Grade GIla. (Silver staining x 625). - Fig. 8. Glandular prostatic carcinoma with high number of AgNORs. Grade GlIb. (Silver staining x 625).
AgNOR pattern of prostatic tissue
Fig. 9. AgNOR pattern of prostatic tissue. Increasing values from normal and BPH tissue to AAH and low grade carcinomas as well as PIN and high grade carcinomas.
Furthermore, in the literature as well as in our own studies, similarities could be demonstrated between AAH and low grade carcinomas as well as between PIN and high grade carcinomas 1, 15, 18,24, 25,38,41,42,43,49,53,61. We have shown that the nucleolar and AgNOR values of atypical adenomatous hyperplasia lie between BPH and low grade carcinomas while the increased nucleolar frequency and mainly eccentric position of the nucleoli and AgNOR number per nucleus of PIN correlate with the value of high grade prostatic carcinomas I4 ,22,25,46,62. In our cases of high grade PIN and AAH with marked architectural and cytological atypias, the basal cell layers were fragmented. Similar to other observations, frequently microglandular and cribriform formations with complete loss of basal cells were in the immediate neighbourhood of high grade PIN8. In such cases with transition of PIN into carcinomas, the nucleolar and AgNOR values of PIN were the same as in high grade carcinomas.
388 . B. Helpap and C. Riede
A close relationship exists between AAH and low grade glandular carcinomas of the transition zone of the prostate concerning architectural/histological, nuclear features (nuclear size variations, chromatin and nucleoli) and the tendency to minimal infiltrative growth pattern. This relationship is also corroborated by the correlation of AAH and low grade carcinomas of 18.2% in our material, with predominance of AAH with marked atypias to carcinomas of 81.8%. In the literature, the coincidence of AAH with prostatic carcinomas ranges between 2.6_22.0%2,11,35. These values and our own results are similar to the reported frequency of incidental glandular carcinoma in the transition zone of the prostate, which ranges from 7-22 %2,23,30. A similarly distinct correlation of PIN to high grade carcinomas exists with a percentage range of 45_97.0%35,59. These values are similar to our own percentage of 70.8% with a predominance of high grade PIN to carcinoma of 85.5%. The preneoplastic character of high grade PIN is also supported by former studies employing autoradiography, DNA cytometry and by analysis of apoptotic bodies. In general, the results obtained with these experimental methods correlate to the AgNOR findings. But we found no differences of AgNOR numbers between basal and luminal cells as described after Ki67/MIB1 and/or PCNA immunohistochemical labeling5,34. AAH and low grade PIN are characterized by euploid DNA values and low labeling indices as well as rare apoptosis which can be mainly observed in well differentiated prostatic carcinomas. Aneuploid DNA pattern and increased numbers of apoptotic bodies, as well as increased labeling indices with tritiated thymidine or by immunohistochemical labeling with Ki67 or PCNA, are typical for hi£h grade PIN as well as highly malignant carcinomas 1, ,4,11,17,21, 26,27,45,47,48,50,54,56,58,60,63,65. In conclusion, we suggest due to topography, architectural and histological disturbances, cytological atypias, immunohistochemical findings with incomplete loss of basal cells cell kinetic findings similar to well and poorly differentiated carcinomas, respectively, that the demonstrated analyses of nucleoli and AgNOR are a further point that prostatic intraepithelial neoplasia may be a true preneoplasia. That means PIN is an accepted precursor of mainly poorly differentiated carcinomas of the dorsoperiphery of the prostate. In contrast, the histological, cytological and cell kinetical features of AAH are intermediate between BPH and low grade carcinoma of the prostate. AAH may be a precursor of transition zone carcinoma but the findings to date are inconclusive. Future follow up studies should therefore address whether the association of AAH and carcinoma is incidental or whether a transition exists from AAH to carcinoma 34 .
Acknowledgement The authors wish to thank Dr. Thomas Hartung and Dr. Bulatko for valuable discussion, statistics and reading the manuscript.
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Received August 3, 1994 . Accepted in revised form February 9, 1995
Key words: Prostatic intraepithelial neoplasia - Atypical adenomatous hyperplasia - Prostatic carcinoma - Nucleoli - AgNOR Prof. Dr. B. Helpap, PO Box 720, D-78207 Singen, Germany, Tel. 07731189300, FAX 07731189411