Zheng-Fen Zhou,  Shi-Zhen Yuan, Associate Professor of Medicine, having 30 papers and books published. Director of the Department of Gastroenterology, the First Affiliated Hospital of Kunming Medical College, and member and Secretary of Yunnan Provincial Gastroenterology Society, won an Advanced Technology & Science Prize, 153 Xichanglu. Kunming 650031 China.
Shi-Zhen Yuan, Gastroenterology Research Laboratory, Sun Yat Sen Memorial Hospital, Sun Yat Sen University of Medical Sciences, Guangzhou 510120 China.
Presented at the 2nd International Conference of Gastroenteropathy in (Chengdu) in 1993.
Supported by: China Medical Board Grant.
Telephone: +86-781-5324888, Ext 532(Office) 538(Home).
Received: 1995-4-20

Subject headings nucleolus organizer region; colonic neoplasms; prognosis

Zhou ZF, Yuan SZ. Prognostic value of silvera2stained nucleolar organizer regions in colorectal carcinoma.
China Nati J New Gastroenterol,1995;1(1):43-47

Abstract
AIM: Recently AgNOR analysis has been regarded as a useful criterion for tumor diagnosis and research. The purpose of this study was to investigate the prognostic value of AgNOR analysis in colorectal carcinomas.

METHODS: Silver staining technique was applied to paraffin embedded tumor tissue sections of 114 patients with colorectal carcinomas. The number, morphology, size and distribution of AgNOR were counted and analysed.

RESULTS: (1)The number of AgNOR in patients who died within 5 years (mean±SD:8.8±2.3 per nucleus, n=27) was significantly higher than that in those who survived beyond 5 years after establishment of diagnosis (6.3±1.8, n=30, P＜0.001). The number of large sized (＞2μm) and small sized (＜1μm) AgNOR in those who died (mean±SD:85.9±20.7, 661.7±250.5 in one hundred nuclei) was significantly higher than that in those who survived(71.7±27.0, 398.3±225.4, P=0.04, 0.00 respectively). Concentrated type of distribution in those who died (10.2%) was significantly fewer than that in those who survived (31.4%,P=0.00). Mixed type of distribution in those who died (25.7%) was significantly more than that in those who survived (7.1%,P=0.00). (2) The number of Ag NOR also related to other factor that affected prognosis of colorectal carcinoma, such as age, histological type, depth of invasion and metastasis to lymph nodes.

CONCLUSION: The AgNOR analysis is a novel and useful parameter in the assessment of prognosis of colorectal carcinoma.

INTRODUCTION
Nucleolar organizer regions(NOR) are markers of rDNA and rDNA transcription. It can provide information regarding gene regulation and control on cell proliferation and differentiation^［1］, and reflect the biological behavior of cancer cells. Recently, AgNOR analysis has begun to be applied to the research for predicting cancer prognosis, but the results were controversial and so far no study has been performed in Chinese patients. In this study we try to explore the role of AgNOR quantitative analysis in the prediction of colonic cancer prognosis and its relationship with other prognostic factors.

MATERIALS AND METHODS
Selection of specimens
One hundred and fourteen patients with colonic cancers were examined, of which 6 0 were male, 54 female. Among them 11 were in age group of ＜29 (9.7%); 70 in age group of 30-59 (61.4%); and 33 in age group of over 60 (29.0%). In 44 cases cancers were located at sigmoid colon; 18 at ascending colon; 14 at both descending colon and ileocaecum; 9 at transverse colon; 8 at splenic flexur e, and 7 at hepatic flexure of colon. In 57 cases, carcinomas invaded serosa; 27, beyond serosa; 25, invaded deep smooth muscles and 5, mucosa, submucosa and shallow smooth muscles. Histological classification was graded in accordance with the diagnosis standards^［2］ as follows: 86 tubular adenomas (including 29 high, 36 moderate and 21 low differentiation), 6 undifferentiated adenomas, 5 signet ring cell adenomas, 12 mucoids adenomas, 2 papillary adenomas, and 3 polypoid adenomas. According to malignant degree they were classified^［3］as follows: 31 low malignant adenomas (including papillate adenomas, highly differentiated aden omas), 51 moderately malignant adenomas (including polypoid adenomas, moderately differentiated adenomas and mucoid adenomas), 32 highly malignant adenomas (including low differentiated adenomas, undifferentiated adenomas and signet ring adenomas). According to operation findings and pathohistological data from resected specimens, there were 56 cases who had no metastasis, 47 had, and 11 were unclear in the metastasis of lymponodi. Among the cases of lymphonodi metastasis we studied 39 cases of primary and metastatic cancers using the AgNOR technique in identical slides. 57 cases have been followed up over 5 years , among whom 27 died and 30 survived. These preserved specimens of biopsy from patients with colonic cancer were obtained between 1984-1991 from the Department of Pathology, Sun Yat sen Memorial Hospital, Sun Yat Sen Medical University.

Methods
Preparation of AgNOR specimens. Tissues were fixed in 10% formalin solution and processed to paraffin wax. Each paraffin embedded block was cut into two 3μm thick sections, one routinely processed haematoxylin and eosin stains and the other submitted to AgNOR staining according to Ploton's modification one step method^［4］.
      Quantitative analysis. Sections were examined in x20 x40, x100 immersion lens and x100 oil immersion lens. Fields were selected at random. 100 cells were examined continuously. Each nucleus was examined in four respects as follows:①The number of nucleoli: Recording the nucleolar numbers and th e AgNOR numbers besides nucleoli. A nucleolus was defined as an AgNOR dot and th e mean number of AgNOR was calculated. ②Shape: In each specimen, the number of AgNOR dots in 100 nuclei was counted in accordance with the foll owing standards: ① Regular type: the shape of AgNOR dots was circular and the rim was more or less smooth, ②Abnormal type: AgNOR dots were bar shape rhomaus or strange and the diameter was longer than 3μm. ③Size: The size of AgNOR dots of each nucleolus was measured and classfied into 3 groups: Large(2μm), medium (1μm) and small (0-1μm), and in each case 100 nuclei were counted and the mea n was calculated by using C2 2n net type objective ruler (Shanghai Third Optical Instrument Factory) measure size. ④Distribut ion: In each specimen, the distribution of AgNOR dots of 100 nuclei was classifi ed as follows:①Gathered type: The regular dots were gathered at the center of nuclei. Generally, the dots were less than 4 per nucleus. ②Scattered type: The irregular dots scattered in nuclei like satellite, and the number of dots exceeded 5 per nucleus. ③Type of dots inside nucleolus: The different dots were gathered in nucleoli. ④Mixed type: Having the characteristics of two types mentioned above.
      Statistical analysis. Analysis of variance (ANOVA) or KrusKal and Wallis rank sum test (Htest) was employed to detect the differences among groups. Meanwhile, Student＇s t test or Wilcoxon＇s rank sum test was undertaken to compare the differences between two groups. For paired designed data, either paired t test or Wilcoxon＇s signed rank sum test was used.

RESULTS
AgNOR could be clearly recognized as black or brown dots in nuclei or nucleoli after staining.

AgNOR and sex
The mean number of AgNOR was 7.1±2.3 dots per nucleus in male colonic cancer g roup and 6.9±2.1 dots per nucleus in female colonic cancer group (P＞0.05).

AgNOR and age
In the age groups of ＜29, 30-59 and over 60, the mean number of AgNOR was 8.5±2.3, 7.1±2.2, and 6.4±1.8 dots per nucleus respectively. The number of the age group of ＜29 was significantly higher than those of 30-59 and over 60 group (P＜0.05), and there was no difference between the latter two age groups (P＞0.05).

AgNOR and location of cancers
The mean number of AgNOR per nucleus was 6.6±1.8 at sigmoid colon, 7.6±2.2 at descending colon, 7.3±3.0 at splenic flexure of colon, 7.0±2.4 at transverse of colon, 6.7±1.3 at hepatic flexure of colon, 6.7±2.1 at ascending colon, and 8.5±2.9 at ileocaecum. There was no significant difference between these groups.

AgNOR and infiltrative degree
The mean number of AgNOR of the colonic cancers which had invaded different layers was as follows: mucosa 5.6±0.6, submucosa and shallow smooth muscle, 5.3±0.9, deep smooth muscle, 7.4±1.8, serosa, and 8.2±2.9 in outer serosa. The number of AgNOR in which cancer had invaded mucosa, submucosa and shallow smooth muscles was less than that in which cancer had invaded serosa and outer serosa (P＜0.05 for both). In comparison of that in the deep smooth muscle group with that in serosa and outer serosa groups, the differences were significant (P=0.00 for both), but no difference was found among other groups(P＞0.05).

AgNOR and histological types
Different types of colonic cancers: The mean number of AgNOR per nucleus was 6.6±1.9 in tubular adenomas, 6.5±0.9 in mucous adenomas, 11.9±1.3 in undifferentiated adenomas, 10.5±1.1 in signet ring cell adenomas, 5.7±0.5 in polypous adenomas and 6.3±0.2 in papillate adenomas The differences of AgNOR number per nucleus were significant in comparison of tubular adenomas with signet ring cell adenomas and undifferentiated adenomas group respectively (both P＜0.01); mucosa adenomas with signet ring cell adenomas and undifferentiated adenomas. No difference was seen among other groups. For different degree of malignancy of colonic cancer, the mean number of AgNOR per nucleus was 52±0.9 in low malignancy, 65±12 in moderate malignancy and 9.3±2.5 in high malignancy (P＜0.01 for all).

AgNOR and metastasis of lymphonodi
In the metastasis of lymphonodi positive group, the number of AgNOR per nucleus was more than that of the lymphonodi metastasis negative group (7.6±2.1 vs 6.4±2.0 P=0.00), and the AgNOR of the lymphonodi metastatic cancers were more than those of the cancers in situ (8.1±2.1 vs 7.3±1.9, P=0.00).

AgNOR and clinical prognosis
The mean number of AgNOR in died group (8.8±2.3) was significantly higher than that in survived group (P=0.00), and there was no overlap bet ween the range of AgNOR number in died or survived groups, while the histological type was similar. The size and distribution of AgNOR but not the shape were significantly different between two groups(Table 1-3, Figure 1-4).

Table 1 The number of AgNOR in died and survived groups with different histologic types of colonic cancer mean±SD (range)

^bSurvived group: 1 undifferentiated adenoma, 1 polypous adenoma and 1 signet ring cell adenoma. Died group: 2 undifferentiated adenomas and 1 signet ring cell adenoma. ^ct=4.5115, P=0.00, between two groups.

Table 2 The number of abnormal shape and giant AgNOR in died and survived groups (mean±SD/100 nuclei)

^a1t=1.7968, P=0.08; ^a2t=1.8494, P=0.0698, vs died group

Table 3 The size of AgNOR in died and survived groupsZ (mean±SD/100 nuclei)

^at=1.6347, P=0.1078; ^bt=2.0958, P=0.0407; ^ct=4.1973, P=0.0001; vs,survived group.

Figure 1 Moderately differentiated tubular adenomas (alive case) 10×20, 10×100)
Figure 2 Moderately differentiated tubular adenomas (death case). The AgNOR numbers, abnormal dots, large and small dots and scattered and mixted types are significantly increased compared with alive cases.(10×40, 10×100).
Figure 3 Moderately differentiated tubular adenomas (primary cancer of colon): 10×20, 10×100).
Figure 4 Moderately differentiated tubular adenomas (lymphonodic metastasis). Compared with the primary cancer, the AgNOR number, large and small dots and scattered type are increased significantly (10×10,10×100)

DISCUSSION
In recent years, there have been substantial studies on the relationship between AgNOR and prognosis of cancers. Several studies on cancers of digestive system suggested that the number of AgNOR in died group was significantly higher than that in survived group^［5］, and that the number of AgNOR increased in the cases of invasion and metastasis of cancer cells^［6］. Griffiths^［7］ observed 100 cases of rectum adenomas and considered that there was no relation between AgNOR numbers and prognosis, cell proliferation and cell DNA ploidy. Liu et al^［8］. found that the number of AgNOR was significantly different between died group and survived group in non Hodgkin's lymphoma, but there were overlaps between two groups in a few cases. Eusebi et al^［9］. found that the mean areas of AgNOR from breast cancer patients who survived short term (＜34 months) were larger than those who survived long term(＞3 years) and no overlaps were observed between the two groups. Therefore, there were controversial results in the AgNOR studies of cancer prognosis.
      In this study we used AgNOR quantitative analysis to access the prognosis of colonic cancer. The results showed: ①the relationship between AgNOR and clinical prognosis was of value in clinical practice: Compared with that in survived group, the number of AgNOR in died group was significantly higher; meanwhile, the large and small dots increased; the distance between two polarities was increased, with the increase of small dots being most significant; the distribution of gathered type decreased while the distribution of mixed type increased. These results suggested that besides the difference in numbers, the size and distribution of AgNOR were significantly different in two groups, and there was no overlap in the range of AgNOR numbers between died and survived groups while histological type was identical. These results were in accord with Eusebi's report. ②the relationship between AgNOR and correlative factors of colonic cancer prognosis: Some studies suggested that the histological types of colonic cancers played a role in the survival rate. Our data showed that the number of AgNOR was significantly different in different histological types of colonic cancers, and the number of AgNOR increased with the degree of malignancy. These results indicated that quantitive analysis of AgNOR could reflect the cell proliferation, differentiation and degree of malignancy and that clinical course process correlated well with survival.
      Depth of invasion was one of the most important factors in colonic cancer prognosis, and the prognosis was significantly different between the groups with shallow invasion and those with deep muscle, serosal or beyond serosal invasion. Our study showed that the number of AgNOR per nucleus was significantly higher in the latter than that in the former. These results also suggested that the number of AgNOR correlated with the prognosis.
      Lymph node metastasis. Lymph node metastasis was an important factor in the prognosis of colonic cancers. Bockmuhl et al^［10］. found that the numbers of AgNOR in breast cancer with lymph node metastasis was higher than that in breast cancer without lymph node metastasis. Kakeji et al^［11］. also found that the number of AgNOR increased in gastric cancer when lymph node metastasis occurred. Our study showed that the number of AgNOR in lymph node metastasis positive group was higher than that in lymph node metastasis negative group. The increase of AgNOR number in infiltrative and metastatic cancer cells suggested that those cells were more biologically active, and the rRNA gene duplication and transcription activity was much stronger. Therefore, the count of AgNOR number contributed to the prediction of cancer cell invasion, metastasis and relapse after operation. We also found the AgNOR number of lymph node metastatic cancer was higher than that of primary cancer of colon. The results were consistent with Ohno's^［12］ results in the lung cancer with metastasis of cartilaginous sarcoma. The phenomenon might correlate with the aberrance of cancer. More efforts are needed to explore the mechanism in colonic cancers. The prognosis of cancers located at rectum was poorer than that located at colon, and the prognosis of cancers in ileocecum and ascending colon was better than that in the other parts of colon. No difference among other sites of colon was found^［3］. However, our study showed that the number of AgNOR was not different in any location of colonic cancers. These results suggested that the number of AgNOR was not correlated with the site of colonic cancers.As to age, in the group below 30 years the prognosis of colonic cancers was poorer than that in 30-59 years and over 60 years groups^［3］. Our study showed that in below 30 years group, the number of AgNOR was higher than that in 30-59 years and over 60 years groups. It suggested that the number of AgNOR increased in the young people who had poorer prognosis.
      AgNOR is a marker of cell proliferation and rRNA transcription. Therefore, cancers with higher number of AgNOR have more active proliferation of cells. These cancers are exuberant, with more biological activity. The patient＇s condition advanced faster and the prognosis of them was worse. In contrast, in those cancer with lower AgNOR number the proliferation of cells was relatively slow with good biological activity. The patients' condition was stable, and the prognosis of patients was good. Moran^［5］studied the prognosis of advanced colonic cancers, and deemed that the traditional clinical and pathological indexes were too difficult to use in prediction of prognosis of colonic cancers. However, AgNOR were considered as reliable prognostic indexes. Ofner^［13］studied the AgNOR and several other prognostic indexes of colonic cancer in post operation patients and found that the value of AgNOR for predicting the prognosis of colonic cancers was more reliable and more accurate than that of the WHO classification system (UIC dividing terms, Jass classification and Duke's classification). Our stud y showed that AgNOR correlated well with clinical prognosis, age, histological types, the degrees of invasion and lymphonodi metastasis. Between died and survived groups with identical histological types no overlaps were observed. As shown in the present study, AgNOR quantitative analysis is a new, useful and reliable index for predicting the prognosis of colonic cancers. Especially, it can be used for the pathologic diagnoses before and after operation of colonic cancers. This analysis can also be used to direct the treatment, and to predict the metastasis and the relapse of cancers. There are broad prospects for applying the AgNOR quantitative analysis to clinical practice.

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