Argyrophilic nucleolar organizer regions in human adrenocortical neoplasms
Yoshiki Kida, Yasuo Takano, and Masahiko Okudaira
Department of Pathology, School of Medicine, Kitasato University, Kanagawa, Japan
Received 2 April 1992/Accepted 2 June 1992
Summary. Argyrophilic nucleolar organizer regions (AgNOR) in human adrenocortical neoplasms, including five carcinomas and ten adenomas, were studied using a semi-automatic image analyzer. Both the number and to- tal area of AgNOR per nucleus in the carcinomas were found to be statistically greater than in adenomas and control tissues. However, there were no statistically sig- nificant differences in total AgNOR area per nuclear area or in the mean area of individual AgNOR dots. The AgNOR of neoplastic and normal cells were of four mor- phological types: type 1 had a few dots at the periphery of the nucleus, type 2 a few dots at the center, type 3 a large round dot along with several small ones at the center, and type 4 numerous diffusely distributed poly- morphic dots. Most type 3 and 4 cells were found in car- cinoma cases. Type 1 cells decreased in proportion to the severity of biological malignancy. It follows from these findings that careful observation of AgNOR should facilitate the distinction of malignant from benign ad- renocortical neoplasms.
Key words: Adrenocortical neoplasms - Nucleolar orga- nizer regions - Image analysis - Morphology
Introduction
Nucleolar organizer regions (NOR) are DNA loops in in- terphase nucleoli and the sites of ribosomal RNA (rRNA) gene clusters. In humans and chimpanzees, NOR are located on the short-arm ends of five acrocen- tric chromosomes (nos. 13, 14, 15, 21, and 22) (Hender- son et al. 1972) and regulate the formation of 18S and 28S rRNA (Evans et al. 1974). These rRNA species are essen- tial to general protein synthesis and are closely related to
cell proliferation (Hall and Levison 1990; Quinn and Wright 1990). Some specific proteins, including the large subunit of RNA polymerase I (Williams et al. 1982), C23 (nucleolin, 110 kDa) (Ploton et al. 1986; Ochs and Busch 1984) and B23 (numatrin or NO38, 78-kDa phosphopro- tein) (Hernandez-Verdun 1983), can be visualized by ar- gyrophilic staining (AgNOR technique) (Howell et al. 1975; Howell and Black 1980). The results of early re- search indicate AgNOR to be possible indicators of pro- liferative ability and recent onco-pathological findings show the number of AgNOR in tumor cells may be cor- related with the grade of biological malignancy (Ploton et al. 1986).
Adrenocortical neoplasms are particularly difficult to diagnose since the biological grade of malignancy does not necessarily correlate with the histological grade of malignancy (Hough et al. 1979; Karakousis et al. 1985; Slooten et al. 1985). Therefore, pathologists are caught in the dilemma of how to distinguish malignant from benign reliably. We therefore investigated the biological nature of adrenocortical neoplasms to ascertain whether AgNOR may be helpful for distinguishing carcinomas from adenomas. There have been a few reports research- ing adrenocortical neoplasms by AgNOR staining: Sasano et al. (1990) reported that the number of AgNOR was greater but without clear difference between carcino- mas and adenomas. Our results showed the opposite.
The present study was carried out to elucidate the bi- ological nature of adrenocortical tumors in terms of AgNOR, especially their morphology, using a semi-auto- matic image analyzer. It is expected that the results will help to clarify some features of adrenocortical tumors since an approach involving AgNOR is presented here for the first time.
Materials and methods
Materials. Twenty cases were examined from the patient files of Ki- tasato University Hospital and the Medical Center Hospital of the Kitasato Institute from 1986 to 1990. They comprised 5 normal ad- renal cortices (5 autopsy cases), 10 adrenocortical adenomas (10 sur-
Type 1
Type 2.
Type 3
Type 4
gical cases) and 5 adrenocortical carcinomas (3 autopsy and 2 sur- gical cases). Histopathological diagnosis was performed according to the criteria of Weiss (Weiss et al. 1989). All adrenocortical carci- nomas demonstrated visceral metastasis.
Staining method. Routinely formalin-fixed (over 12 h) and paraffin- embedded tissue was sectioned at 3 um and mounted on slides coated with 2% gelatin. Extensive washing with deionized water was done after deparaffinization. The AgNOR staining solution was prepared according to the method of Crocker and Nar (1987) by dis- solving gelatin in 1% aqueous formic acid at a concentration of 2%. It was then mixed 1:2 by volume with 50% aqueous silver nitrate solution to make the final working solution and poured over the tis- sue sections, which were left for 25-30 min at 25° C in a dark room. After washing off excess colloidal silver solution with deionized water, the sections were dehydrated to xylene and mounted in a syn- thetic medium. Routine hematoxylin/eosin-stained serial specimens were also prepared.
Counting procedure. Only extra-nucleolar AgNOR and single-dot nucleoli were counted in the present study. Seven to ten fields per section were selected at random, photographed at × 625 through a Leitz Orthoplan and blown up to a final magnification of x 4200. A sample of 100 nuclei per case, selected randomly, were examined for (a) AgNOR number, (b) total AgNOR area, (c) ratio of total AgNOR area per nuclear area, (d) mean area per AgNOR dot and (e) nuclear area. All these parameters were examined in the same cells. Total AgNOR area and nuclear area were measured three times using a semi-automatic image analyzer (Zeiss, IBAS1, FRG) and the mean values were used. In normal adrenal cortices, mea- sured values were obtained for AgNOR in the zona glomerulosa, zona fasciculata and zona reticularis cells separately, and then aver- aged to obtain the mean values. In the adrenocortical adenomas, measured AgNOR values were obtained from areas occupied by clear cells.
Statistical analysis. Comparison of each of the measured parameters for AgNOR in the three groups was made with one-way analysis of variance by the Kruskal-Wallis rank-sum test and comparisons of two of three groups by the Wilcoxon-Mann-Whitney test. All deter- minations were made using a computer statistics package (FISHER, Version 1.2, Nakayama Inc, Tokyo, Japan).
Morphological classification. AgNOR dots in adrenocortical tumor cell nuclei were morphologically classified into the following four groups based on number, shape and location (Fig. 1): type 1, a few uniform and round-shaped AgNOR dots at the periphery of the nucleus with at least one attached to the inner nuclear rim; type 2, AgNOR dots somewhat larger than type 1 and uniform in size and round in shape, but located at the center of the nucleus and without inner nuclear rim contact; type 3, one or a few, large and round- shaped AgNOR dots along with one to several smaller ones sited at the nuclear center; type 4, AgNOR dots of polymorphic shape and size distributed diffusely throughout the nucleus. Intermediate cell types were rare in this study.
Results
Clinico-pathological evaluation
The results of clinico-pathological evaluation of the 20 cases are summarized in Table 1. Carcinoma weights ranged from 70.0 g to 1670.0 g, with a mean of 788.0 g. Sizes were from 8.0 cm to 16.0 cm for the greatest diam- eter with a mean of 12.6 cm. For adrenocortical adeno- mas, these values were 11.6 g and 2.5 cm respectively. There were no adenomas over 30.0 g in weight or 4.0 cm in size. Clinical findings regarding hormonal function in- dicated Cushing’s ‘syndrome for 3 of the 5 carcinomas and 5 of the 10 adrenocortical adenomas. Conn’s syn- drome was evident in 4 of the 10 adrenocortical adenoma cases. No clinical adrenocortical hormonal abnormality could be detected in any normal adrenal cortex.
Analysis of AgNOR and nucleus
AgNOR dots were observed in the nuclei to be black- brown argyrophil spots of various sizes and shapes, in all cases examined (Figs. 2, 3).
AgNOR numbers. The mean number of AgNOR in the carcinomas was 2.50±0.68 (mean+SD), in adenomas 1.55±0.23, and in normal controls, 1.53±0.28 (Fig.4). In the adenomas and controls, one to three AgNOR dots per nucleus were present with no significant differences in size or shape. The mean number of AgNOR in cortical carcinomas was greater than in adenomas and normal controls. Statistically significant differences were found between carcinomas and adenomas (P=0.0013), and be- tween carcinomas and normal controls (P=0.0159), but not between adenomas and normal controls. Overall comparison showed statistical significance (P=0.0107).
Mean AgNOR total area per nucleus. The average total area of AgNOR was 9.24+4.42 um2 in carcinomas, 5.27±0.48 um2 in adenomas, and 4.46±0.69 um2 in the controls (Fig. 5). This parameter increased with histo- pathological grading. Differences were statistically sig- nificant for carcinomas and controls (P=0.0079), adeno- mas and controls (P=0.0400), and carcinomas and adenomas (P<0.0001). Overall, a statistically significant difference was evident (P=0.0014).
Mean nuclear area. The average nuclear area was 50.61 ±11.47 pm2 in carcinomas, 34.28+2.76 um2 in
| Disease | Case | Age (years) | Sex | Clinical syndrome | Meta- stasis | Weight (g) | Size (cm) | Weiss' score |
|---|---|---|---|---|---|---|---|---|
| Carcinomas | C1 | 24.0 | F | Cushing synd. | (+) | 700.0 | 14.0 | 9.0 |
| C2 | 71.0 | M | Cushing synd. | (+) | 300.0 | 10.0 | 9.0 | |
| C3 | 35.0 | F | Cushing synd. | (+) | 70.0 | 8.0 | 7.0 | |
| C4 | 37.0 | M | (-) | (+) | 1670.0 | 16.0 | 9.0 | |
| C5 | 56.0 | F | (-) | (+) | 1200.0 | 15.0 | 9.0 | |
| Mean | 44.6 | 788.0 | 12.6 | 8.6 | ||||
| Adenomas | A1 | 33.0 | F | Cushing synd. | (-) | 16.0 | 3.0 | 0.0 |
| A2 | 38.0 | F | Conn synd. | (-) | 6.0 | 2.0 | 0.0 | |
| A3 | 30.0 | F | Cushing synd. | (-) | 18.0 | 4.0 | 4.0 | |
| A4 | 40.0 | F | Conn synd. | (-) | 12.0 | 1.3 | 0.0 | |
| A5 | 36.0 | F | Cushing synd. | (-) | 7.5 | 2.8 | 1.0 | |
| A6 | 64.0 | F | Cushing synd. | (-) | 30.0 | 3.5 | 2.0 | |
| A7 | 38.0 | F | Conn synd. | (-) | 5.8 | 1.8 | 0.0 | |
| A8 | 67.0 | M | (-) | (-) | 8.2 | 2.0 | 1.0 | |
| A9 | 49.0 | M | Conn synd. | (-) | 2.2 | 1.5 | 0.0 | |
| A10 | 24.0 | F | Cushing synd. | (-) | 10.5 | 3.4 | 2.0 | |
| Mean | 41.9 | 11.6 | 2.5 | 1.0 | ||||
| Normal | N1 | 46.0 | M | |||||
| controls | N2 | 43.0 | F | |||||
| N3 | 55.0 | M | ||||||
| N4 | 45.0 | F | ||||||
| N5 | 53.0 | F | ||||||
| Mean | 48.4 |
2a
2b
adenomas, and 31.52+4.37 um2 in the controls. Differ- ences were statistically significant for carcinomas and adenomas (P=0.0013), and carcinomas and the controls (P=0.0159). Overall, a statistically significant difference was evident (P=0.0052).
Total AgNOR area per nuclear area. The mean ratio of total AgNOR area to the nuclear area was 0.19 ± 0.06 for carcinomas, 0.16±0.01 for adenomas, and 0.14±0.01 for the controls, the values being essentially similar.
Mean area per AgNOR dot. The mean area of single AgNOR dots was 4.29±1.05 um2 for carcinomas, 3.70±0.47 um2 for adenomas, and 3.21±0.66 um2 for the controls.
Morphological classification of AgNORs
All cells examined were typed using the proposed mor- phological classification of nuclear AgNOR dots. As summarized in Fig. 6, type 1 predominated, at a rate of
3a
3b
*
4
**
Number of AgNORs per nucleus
w
. …
2
-
. … .
1
T
1
CONTROLS
ADENOMAS
CARCINOMAS
Fig. 4. Scattergrams illustrating the results for mean numbers of AgNOR per nucleus. Comparisons between normal controls and carcinomas, and adenomas and carcinomas revealed significant dif- ferences: * P=0.0159; ** P=0.0013. O, Mean; vertical bars, SD
more than 50% in all adenoma and control cases. On the other hand, the incidence of type 1 was less than 30% in carcinomas. Furthermore the majority of types 3 and 4 were found in carcinomas.
Discussion
In the present study, the number of AgNOR in ad- renocortical neoplasms was found to increase in propor- tion to the grade of malignancy with statistical signifi- cance. In other malignancies, such as lymphomas (Crocker and Nar 1987), breast cancer (Ruschoff et al.
*
**
17
Total area of AgNORs per nucleus (um-)
L
10
9
8
…
7
6
-
·
5
…
I
1
4
3
CONTROLS
ADENOMAS
CARCINOMAS
1990), renal cell carcinoma (Delahunt et al. 1991) and meningeal tumors (Chin and Hilton 1991), AgNOR numbers in tumor cells have similarly been reported to correlate well with the grade of malignancy. On the other hand, some authors have described the value of AgNOR numbers in grading of malignancy to be limited in lung cancer (Soomro et al. 1991), thyroid cancer (Nairn et al. 1988) and soft-tissue sarcoma (Wrba et al. 1991).
While our results were positive, Sasano et al. (1990) demonstrated that AgNOR numbers were of little value in discerning malignancy in adrenocortical neoplasms, but rather were correlated with increased steroid hor-
(%)
100
TYPE 4
TYPE 3
TYPE 2
TYPE 1
50
0
CONTROLS
ADENOMAS
CARCINOMAS
mone production. This discrepancy may be due to the complexity of adrenocortical neoplasms and the number of cases examined. In addition, differences in the count- ing method may be the underlying reason. There are two approaches to counting AgNORs (Orrell et al. 1991). In silver-stained preparations, dots are apparent both out- side and inside the nucleolus. Some authors estimate only extra-nucleolar AgNOR and count the nucleolus as a sin- gle dot, whilst others count both extra- and intra-nucleo- lar AgNOR. Crocker et al. (1989) have proposed a stan- dardized approach of counting both intra- and extra-nu- cleolar AgNOR, which serves well for distinguishing ma- lignant lesions from benign breast neoplasms. Orrell et al. (1991) showed that counting both intra- and extra-nu- cleolar AgNOR facilitated the distinction of benign melano-naevi from malignant melanoma more effec- tively than counting that excluded intra-nucleolar AgNOR. If all AgNOR (including those within the nucleolus) are counted, the mean numbers clearly differ and this would thus appear a better method. However, for adrenocortical tissue, application of this method would be difficult. Thus, as far as we know, intra-nucleo- lar AgNOR cannot be accurately counted for some human tissues even with careful staining, since there are malignant neoplasms for which aggregation or homoge- nization of intra-nucleolar AgNOR is difficult to demonstrate with increasing malignancy. Even at maxi- mum magnification (employing × 100 oil objective), ac- curate counting is difficult in the case of many intra-nu- cleolar AgNOR. Thus, in the present study, the nucleolus was counted as a single dot.
For accurate estimation of AgNOR dots differing in size, the mean total area of AgNOR per nucleus (mean AgNOR area) was also determined with a semi-auto- matic image analyzer. The results demonstrated that this parameter might be particularly useful for differentiating adrenocortical carcinomas from adenomas and normal tissue. Another recent report showed a significant corre- lation between the quantity of AgNOR (mean AgNOR area) and cell doubling time in 13 neuroblastoma cell lines (Trere et al. 1989). Furthermore a significant rela- tion between the size of AgNOR and the malignancy grade of non-Hodgkin lymphomas was established (Crocker and Egan 1988).
The mean ratio of total AgNOR area per nuclear area and mean area per AgNOR dot were shown in this study not to be useful for distinguishing carcinomas from be- nign lesions. That the mean nuclear dimensions and mean AgNOR number increase with mean AgNOR area may possibly be the reason for this (Spearman’s correla- tion coefficient = 0.8045/0.6947, P<0.0001/0.0007 re- spectively).
Differences in AgNOR size and shape may exist be- tween malignant and benign lesions (Crocker and Egan 1988) although the morphology of AgNOR has yet to be clarified in detail. The intra-nuclear location of AgNOR and the combination of AgNOR dot sizes were given par- ticular attention in this study and, using the present mode of classification, carcinomas, adenomas and controls could be clearly distinguished from each other. Nearly all AgNOR in carcinoma cells were found at the center or scattered throughout the nucleus. The percentage of type 1 at the periphery decreased with the degree of bio- logical malignancy. Types 3 and 4 AgNOR, by contrast, were found primarily in adrenocortical carcinomas, this appearing a most remarkable difference between benign and malignant lesions.
Various attempts have been made to assess the malig- nancy of adrenocortical neoplasms. Immunohistochemi- cal studies on adrenocortical tumors have indicated the vimentin antigen to be present only in carcinomas, but not in adenomas or the normal adrenal cortex (Miettinen et al. 1985). However, this is disputed by some investiga- tors, who have found malignant and benign adrenocorti- cal tumor cells to express positive reactions for anti-vi- mentin antibody (Wick et al. 1986). Lectins are consid- ered useful markers for evaluating malignant trans- formation in miscellaneous human tissues (Cooper 1984) but, in the case of adrenocortical tissue, they cannot be used to differentiate benign from malignant lesions (Sasano et al. 1989). DNA ploidy analysis by flow cyto- metry appears to hold promise for this purpose (Bowlby et al. 1986; Klein et al. 1985), but no significant correla- tion between DNA ploidy pattern and biological behav- ior has yet been shown (Camuto et al. 1987; Cibas et al. 1990).
In conclusion the determination of mean AgNOR number and area may, in conjunction with the AgNOR
morphological classification, facilitate the distinction of benign from malignant adrenocortical neoplasms. The shape and location of AgNOR were shown to have par- ticular application as possible indicators of physiological differences.
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