A clinicopathological evaluation of nucleolar organizer region proteins in human breast carcinoma

Surgical Oncology 1994 ; 3 :

53-57

A clinicopathological evaluation of nucleolar organizer region proteins in human breast carcinoma Y . YOSHIDA, T . OKAMURA, K . YANO, M . KODATE, T . OYAMA, K . INUTSUKA, S . TAGA AND T. EZAKI

Second Department of Surgery, School of Medicine, University

of

Occupational and Environmental Health, Kitakyushu

807,

Japan

The number of nucleolar organizer region proteins (NORs) identified by silver staining (Ag) was studied in 106 primary breast carcinomas. The AgNOR score (mean±SD) in tumours smaller than 2 cm was sinificantly lower than that in tumours larger than 2 cm (P<0.05) . However, there was no significant difference according to other clinicopathological characteristics . According to postoperative distant recurrence, the AgNOR count was significantly higher in the group with recurrence than in the group without recurrence (P<0.05) . Further, the survival rate was significantly lower in the high AgNOR score (? 3) group than in the low score (<3) group (P<0 .01) . The present study indicates that the AgNOR score in human breast cancer cells is useful for evaluating cell proliferative activity and may help predict postoperative distant recurrence and, ultimately, the postoperative prognosis of patients .

Keywords :

Surgical Oncology

1994 ; 3 : 53-58 .

AgNORs, breast carcinoma, clinicopathological study .

INTRODUCTION

In the present study, we investigated the relationship between the numbers of AgNORs and clinicopathological characteristics, postoperative

Nucleolar organizer regions (NORs) are chromosomal segments encoding ribosomal RNA (rRNA)

recurrence in other organs and the prognosis of patients with breast cancer .

and are associated with argyrophilic, acidic, nonhistone proteins . Ribosomal RNA genes ultimately direct ribosome and protein synthesis, and it has been suggested that the number of NORs detected

SUBJECTS AND METHODS

in the cell may reflect nuclear and cellular activity [1] . A recent modification of a silver staining technique (one-step method) allows NORs to be visual-

Clinical tissues

ized in paraffin sections, where they are called argyrophilic nucleolar regions (AgNORs)

We examined 106 patients with primary breast

[2-4] .

carcinoma who had undergone radical mastectomy

Smith and Crocker demonstrated a difference in

and lymph node dissection in the Second Depart-

numbers of AgNORs between benign and malignant

ment of Surgery, University Hospital of Occupational

breast epithelium and proposed that this technique

and Environmental Health, Japan, from 1980 to

might be a valuable adjunct to prognostication in have

1990 . Every resected specimen was routinely fixed in 10% formalin-saline and paraffin embedded .

determined that there is a relationship between the

Macroscopic and microscopic evaluations were made

number of AgNORs and the degree of lymph node

according to the rules established by the Japanese

involvement or tumour growth in breast carcinomas .

Society for Mammary Cancer [9] .

Correspondence :

Staining technique for AgNORs

breast carcinoma

[5] .

Other studies

[6-8]

Y . Yoshida, Second Department of Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan .

Silver NORs (AgNORs) were demonstrated according to the method of Ploton 53

et al. [1] .

The colloidal



54

Y. Yoshida et al .

silver-staining solution was prepared by dissolving 2% gelatin in 1% aqueous formic acid, which was

Statistical analyses

then diluted to a ratio of 1 :2 by volume with 50%

Comparisons of AgNORs counts for each clinico-

aqueous silver nitrate . Three micron-thick paraffin

pathological characteristic and between recurrence-

sections were transferred gradually to water via

positive and -negative groups were made using the

xylene and graded alcohols and then covered with

Student's unpaired t-test and Mann-Whitney U-test

the colloidal silver solution for 40 min in the dark

or the Kruskal-Wallis test . Survival was analysed by

at room temperature (18-20°C) . After thoroughly

the

rinsing with deionized water to remove unwanted

Wilcoxon test was used to compare the survival

silver precipitates, the sections were stained with

curves .

Kaplan-Meier

method .

The

generalized

2% chloroauric acid solution for 5 min . These specimens were then counterstained using Meyer's haematoxyline and dehydrated through graded

RESULTS

ethanol to xylene . The relationship between the clinicopathological Counting procedure

characteristics and the number of AgNORs (mean±SD) is shown in Table 1 . There was no

The number of AgNORs, silver-stained black or dark

statistically significant difference in the AgNOR

brown dots, within the nucleus was counted . For

counts corresponding to patient characteristics such

each specimen, the total count of AgNORs in 100

as age, marital status, menopausal status and

cancer cells, selected from multiple random fields,

location of tumour . Further, there was no significant

was determined under an oil immersion lens at

difference corresponding to pathological charac-

x 1000 by microscopy (BH-2, Olympus, Japan) and the mean number of AgNORs per nucleus was

teristics such as histological type, lymph node involvement and histological stage . However, the

calculated (Figures 1 and 2) .

AgNOR score in tumours smaller than 2 cm was

Figure 1 . Low mean number of AgNORs in a breast carcinoma specimen (silver-colloidal staining, x 1000) .

Nucleolar organizer regions in human breast cancer

55

Figure 2 . High mean number of AgNORs in a breast cancer specimen (silver-colloidal staining, x 1000).

significantly lower than that in tumours larger than 2 cm (P<0 .05) . Although the AgNOR score was lower

colon cancer [13] and oesophageal carcinoma [14] .

the ER-negative group, there was no significant

In breast cancer, previous reports have demonstrated that the AgNOR count is useful in distinguishing benign from malignant lesions and is

difference between the two (Table 2) .

related to the likelihood of metastasis to axillary

in the estrogen receptor (ER)-positive group than in

The mean AgNOR count was compared between

lymph nodes [7, 8] . However, the usefulness of the

two groups, one with the presence and the other

AgNOR score in predicting postoperative recurrence

with the absence of postoperative recurrence to

or prognosis in patients with breast cancer has not

distant organs (Figure 3), except for local (n=4) and

been studied .

lymphatic (n=10) recurrences . The count was

The present study revealed that the AgNOR count

significantly higher in the group with distant recur-

is related to tumour size . Studies of the correlations

rence than in the group without such recurrence

between the AgNOR count and tumour size has had

(P<0,05) .

varied results . Canepa et al. [15] reported that the

The 106 patients were divided into two groups

AgNOR score did not correlate at all with tumour

according to the number of AgNORs as follows :

size, while Mourad et al . [16] and Ohri et al. [17]

low-score group, <3 (75 patients) and high-score group, ? 3 (31 patients) . The survival rate was significantly lower in the high-score group than in the

suggested that there was a positive correlation

low-score group (P<0 .01) (Figure 4) .

revealed that the AgNOR count does not increase as the tumour grows and that the AgNOR score does

between the AgNOR score and the T-stage . Our data support, in part, the latter conclusions . However, we

not increase further once the tumour reaches a size DISCUSSION

of 2 cm . We determined that in breast carcinoma, tumours over 2 cm in size have high malignant

Some reports have proposed that the AgNOR score expresses the malignant activity and is a prognostic determinant in malignant diseases such as lung cancer

[10],

hepatoma

[11],

gastric cancer [12],

activity . There was no significant relationship in the present study between the AgNOR score and the involvement of lymph nodes . However, conclusions



56

Y. Yoshida et al . P< Q05

Table 1 . Relationship between the number of AgNORs and clinicopathological characteristics in breast carcinoma Number of AgNORs (mean ±SD) Age <-39 40-59 ? 60 Marital status Married Single Menopausal status Premenopause Postmenopause Location of tumour Rt breast Lt breast Tumour size (cm) s2 .0 2 .1-5 .0 ? 5 .1 Histological typet Papillo-tubular carcinoma Solid-tubular carcinoma Scirrhous carcinoma Other Lymph nodes metastasis Negative Positive Histological staging 1 II III IV

2 .50±0 .87 2 .69±1 .06 2 .51 ± 0 .80

P-value* 3 .00

NS 0

2 .76±0 .82 2 .73±1 .28

NS

2 .49±0 .91 2 .66±0 .98

NS

2 .47±0 .77 2 .69±1 .06

NS

2 .13±0 .57 2 .74±1 .04 2 .72+0 .89 2 .76±0 .97 2 .48±0 .99 2 .21 ±0 .84 2 .72 ± 0 .91 2 .54±0 .93 2 .70±0 .99 2-55+0 .94 2 .57±0 .94 2 .51 ±1 .00 3 .13±1 .07

3.05+--1 .17 -

Absence Presence ; .a=67! (n=25) Pos'operab.ve d .start recurrences Figure 3 . Scattergram of AgNOR scores according to the presence and absence of postoperative distant recurrence . The mean number of AgNORs in the recurrence positive group was significantly higher than in the group without recurrence (P<0 .05) .

<0 .05 0 NS

NS

NS

*P-value based on Student's unpaired t-test and the Mann-Whitney U-test or the Kruskal-Wallis test . (INS = not significant)l tHistological type determined according to the rules

0 3 a

100 90 80 70 60 50 40 30 20 10 0

AgNOR: < 3 AgNOR :>~3

0

P<001

I 4I Years after surgery

10

Figure 4 . Survival rates of patients with high and low mean AgNOR scores . The rate in the high AgNOR score group was significantly poorer than that in the low AgNOR5 score group (P < 0 .01) .

established by the Japanese Society for Mammary Cancer . Table 2. Hormonal receptor status and AgNOR Hormonal receptor

Estrogen receptor (ER) Positive Negative Progesterone receptor (PgR) Positive Negative

Number of AgNORs (mean ± SD)

P-value*

2 .40±0 .87 2 .81 ± 1 .07

NS 0 .07

2 .31 ±0 .83 2 .69±1 .02

NS

*P-value based on the Mann-Whitney U-test (NS = not significant) .

regarding this relationship have been controversial . Raymond and Leong [7] reported that there was no relationship between the AgNOR count and axillary node status ; however, Lawry et al [18] reported the opposite conclusion . We presume that since breast cancer is a systemic disease, the malignant activity of breast carcinoma cannot be determined exclusively by the presence or absence of axillary lymph node metastasis . One factor which indicates the malignant activity of cancer is the presence of postoperative distant recurrences . In the present study, the AgNOR score of the patient group with postoperative distant recurrence was significantly higher than in the

Y Yoshida et al . group without such a recurrence . This indicated that the AgNOR score in human breast carcinoma may predict postoperative distant recurrences . Furthermore, the survival rate of the high AgNOR score group was poorer than that of the low-score group . The AgNOR score may indicate only cancer cell proliferation at the time of surgery and may not represent the true malignant activity of a tumour . In

57

resolution localisation of nucleolar organizing region protein and nuclear protein in interphase and mitotic nuclei . Histchem J 1984 ; 16 : 897-906 . 5 . Smith R, Crocker J . Evaluation of nucleolar organizer region-associated proteins in breast malignancy . Histopathology 1988 ; 12 : 113-25 . 6 . Dervan P, Gilmartin L, Loftus B, Carney D . Argyrophilic nucleolar organizer region counts correlate with Ki67 scores . Am J Clin Pathol 1989 ; 92 : 401-7 .

practice, however, we have treated patients with

7 . Raymond WA, Leong A . Nucleolar organizer regions

breast carcinoma in various stages and have not

relate to growth fractions in human breast carcinoma .

found definite predictors of postoperative distant recurrence or prognosis . Thus, the AgNOR score may enable us to identify for clinical purposes patients with breast cancer who are at a high risk of distant recurrence . In conclusion, we consider that enumeration of AgNORs in human breast cancer cells provides us with a means of evaluating cell proliferative activity and that the AgNOR score in breast cancer may help predict postoperative recurrence to other organs and, ultimately, the postoperative prognosis of patients.

Hum Pathol 1989; 20 : 741-6 . 8 . Sivridis E, Sims B . Nucleolar organizer regions : new prognostic variable in breast carcinomas . J Clin Pathol 1990 ; 43 : 390-2 . 9 . Japanese Breast Cancer Society . The general rule for clinical and pathological record of breast cancer . Jpn J Surg 1989 ; 19 : 612-32 . 10 . Crocker J, Ayres J, McGovern J . Nucleolar organizer regions in small cell carcinoma of the bronchus . Thorax 1987 ; 42 : 972-5 . 11 . Crocker J, McGovern J . Nucleolar organizer regions in normal, cirrhotic, and carcinomatous livers . J Clin Patho/ 1988 ; 41 : 1044-8 . 12 . Suarez V, Newman J, Crocker J, Collins M . The value of NOR number in neoplastic and non-neoplastic

ACKNOWLEDGEMENTS

This work was supported, in part, by the Grant-in-

epithelium of the stomach . Histopatho/ogy 1989 ; 14 : 61-6 . 13 . Ofner D, Totsch M, Sandbichler P et al. Silver stained

Aid, 1991, from the Fukuoka Cancer Society, Japan .

nucleolar organizer region proteins (Ag-NORs) as a

We wish to express our gratitude to Dr Tetsuo

predictor of prognosis in colonic cancer . 1990 ; 162 : 43-9 .

Hamada and Mr Hitoshi Fujiwara, Department of Pathology, University of Occupational and Environmental Health, Japan, for their technical advice on pathological staining .

J Pathol

14 . Morita M, Kuwano H, Matsuda H, Moriguchi S, Sugimachi K . Prognostic significance of argyrophilic nucleolar organizer regions in esophageal carcinomaCancer Res 1991 ; 51 : 5339-41 . 15 . Canepa M, Gambini C, Sementa AR, Borgiani L, Rovida S . Nucleolar organizer regions and Ki-67 immunostain-

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