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1 Title Author(s) Citation Kobe University Repository : Kernel Issue date Resource Type Resource Version DOI URL Argyrophilic Nucleolar Organizer Regions (AgNOR) in Colorectal Cancer Patients : Correlation with Survival Tabuchi, Yoshiki / Nakamura, Takeshi / Nakae, Shiro Bulletin of allied medical sciences Kobe : BAMS (Kobe),12:83-87 Departmental Bulletin Paper / 紀要論文 publisher Create Date:

2 Argyrophilic Nucleolar Organizer Regions (AgNOR) in Colorectal Cancer Patients: Correlation with Survival Yoshiki Tabuchi 1, Takeshi Nakamura 2 and Shiro Nakae 2 The score of argyrophilic nocleolar organizer regions (AgNOR) was determined in 80 colorectal cancer patients, and the correlation between AgNOR score (dot number/nucleus) and survival was examined. The survival curve in 40 patients with low AgNOR score under the mean (3.81) of all the primary cancer lesions was significantly better than that in 40 patients with high AgNOR score over the mean, and the 4-year survival rate was 94% in the former and 71 % in the latter. Significant differences between both patient-groups were found in 6 out of 11 background variables: deep cancer invasion, node and liver metastases, Dukes stage D, low grades of node dissection and curability C were significantly more in the patients with high AgNOR score than in the patients with low AgNOR score. These results suggest that the determination of AgNOR score in the cancer lesions is clinically used as a prognostic parameter in colorectal cancer but that the score is not an independent parameter from the clinicopathologic prognostic parameters. Key Words Colorectal cancer, Argyrophilic nucleolar organizer regions (AgNOR), Clinicopathologic variables, Prognosis. INTRODUCTION Nucleolar organizer regions (NOR) are loops of ribosomal DNA (r-dna) encoded for r-rna production 1. 2 ). Proteins associated with the NOR are called AgNOR proteins, which are microscopically visualized as black dots in the sections stained with silver colloidal solution 3-5 ). Recently, close relationships between AgNOR Faculty of Health Science l, and First Department of Surgerl, Kobe University School of Medicine, Kobe Japan. dot number or score and parameters of proliferative activity of cells have been reported 4-6 ). At the present time, the measurement of AgNOR is generally thought to be a simple method for the estimation of cellular proliferation activity, and an increase of AgNOR proteins has been reported in a variety of malignant tumors:i.7-16). However, the clinical value remains a matter of controversy, because the antagonistic results have been reported 7-16 ). Thus, it has been vigorously examined now whether the clinical value of AgNOR determination is present. In the present study, AgNOR score was determined in 80 colorectal cancer patients, and the correlation of the score with survival was examined, in order to clarify the clinical value of AgNOR determination in colorectal cancer. Vol. 12, 1996 Bulletin of Allied Medical Sciences, Kobe 83

3 Y. Tabuchi et al. MATERIALS AND METHODS Eighty patients with primary colorectal adenocarcinoma, who underwent operative resection of the cancer lesions during 4 years from 1991 to 1994 in the First Department Surgery, Kobe University Hospital (Kobe, Japan), were included in this study. Patients treated preoperatively cytotoxic agents were excluded, because the agents affect the AgNOR score which represents cellular proliferation activity. The surgically resected specimens were fixed in 10% neutral formalin solution, and the specimens were fixed within 48 hours to avoid the reduction of AgNOR staining 20 ). U sing serial 3,um sections in parallel with the histological observation sections stained with hematoxylin and eosin, AgNOR was stained according to Crocker and co-workers 3 ). In brief, Fgiure 1. AgNOR is found as black dots with various forms and sizes in cancer nuclei. AgNOR stain, Original mag nification X 200. ctl >.~ 60 ::J (J) 100 -r l...l..i...i...l.l...r..,,, t..., L..... l: Ull :lll 1 71 %.... a.11.t.ll.l. P<O.01 (z=2.83) -- : low AgNOR group (n=40)... : high AgNOR group (n=40) a Postoperative period (year) Fgiure 2. Survival curves and the 4- year survival rates of the low and high AgNOR groups. the staining solution which consisted of 1 volume of 2 % gelatin in 1 % formic acid and 2 volume of a silver nitrate solution was pored over the deparaffinized sections, and the preparations were left in the dark for 40 minutes at 40 C. The silver solution was washed off using deionized water, and the sections were dehydrated to xylene and mounted on slide glass. After color photography of the most deeply invasive parts in the cancer lesions was taken at the original magnification X 200, the number of AgNOR black dots was counted on the trebly enlarged photographs (Fig. 1). Two hundred nuclei in each lesion were analyzed, and the mean number of the dots per nucleus was defined as AgNOR score for each lesion. Survival curves and rates of the patients were estimated by Kaplan-Meir method. Eleven clinicopathologic variables consisting of age, sex, tumor 84 Bulletin of Allied Medical Sciences, Kobe

4 AgNOR score in colorectal cancer patients size, location, microscopic type, depth of cancer invasion, node and liver metastases, Dukes stage, grades of node dissection and curability were examined as the background variables. Almost all of the variables were recorded according to the "General Rules for Clinical and Pathological Studies on Cancer of the Colon, Rectum and Anus,,18,19). Stage classification was determined by Dukes classification 20 ) which was one of the worldwide available staging systems of colorectal cancer. The statistical difference of survival curves was determined by the generalized Wilcoxon test. Differences of the mean AgNOR score in the cancer lesions and distribution of the patient-number were statistically analyzed by the Student's t test and chi-square test or Fisher's exact probability calculation test, respectively. RESULTS 1. Survival curves and rates due to the difference of AgNOR score The mean of AgNOR score in all the cancer lesions was In the present study, the patients were classified into 2 groups, using the mean of all the lesions: 40 patients with high AgNOR score over the median (high AgNOR group) and 40 patients with low AgNOR score under the median (low AgNOR group), and the survival curves and rates were examined between 2 groups. The survival curve was signfifcantly better in the high AgNOR group than in the low AgNOR group, and the 4-year survival rate was 94, % in the latter and 71 % in the former (Fig. 2). 2. Background variables in the pa- Table 1. Clinicopathologic background variables in the low and high AgNOR groups Clinicopathologic Low AgNOR High AgNOR variable (n=40) (n=40) Age t (year) 66.2± ±9.7 Sex male female Tumor size t (mm) 50.9± ±23.2 Location colon rectum Microscopic well type mod por. 4 6 Depth of m-pm 15 6 invasion ss(alfsi(ai) 25 34** Liver negative metastasis positive 3 11* Node negative metastasis positive 13 23** Dukes stage Node dissection A-B C D 3 11** 1 2 8** 2 2 5** A Curability B 7 2 C 2 8** t Mean ± standard deviation (S.D.) * Significant (p< 0.01) different from low AgNOR. **Significant (p<0.05) different from low AgNOR. tients with high and low AgNOR scores As shown in Table 2, advanced cancer lesions with deep cancer invasion, node and liver metastases and with Dukes stage C, low grade of node dissection and curability C were signficantly more in the high AgNOR group than in the low AgNOR group (Table 1). Vol. 12,

5 Y. Tabuchi et al. DISCUSS~ON In general, the measurement of AgNOR score is thought to be a simple method for the estimation of cellular proliferation acti vit/- 6 ). The clinical value of AgNOR determination is vigorously examined in a variety of hyperplastic and neoplastic lesions, and an increase of AgNOR number or score has been reported in a variety of cancers ). However, the clinical value has remained unclear, because antagonistic results have been reported ). Thus, it has recently been a matter of controversy whether or not the clinical value of AgNOR determination in cancer lesions is present. In order to clarify the clinical value, the relation of AgNOR score to survival was examined in colorectal adenocarcinoma by the determination of AgNOR score in the most deeply invasive part of every lesion, because the proliferative activity of cancer cells is well-known to be different from the examined parts in an identical tumor. In this study, in was confirmed that the survival curve of the low AgNOR group was significantly better than that of the high AgNOR group, as already reported by some investigators ). Thus, it is concluded that the determination of AgNOR score is clinically used as one of the prognostic parameters, at least, in colorectal cancer. On the other hand, some investigators have re- ported no or little clinical value of AgNOR determination in various malignant tumors9.10.u.14). The causes are unclear. However, one of the causes may be due to the inappropriate fixation time and/or methods of the specimens for the determination of AgNOR score, as already reported by Griffinth and co-workers 20 ). The causes may be based on the stage and/ or kind of tumors, as suggested by the some investigators ). The examination of the clinicopathologic background variables revealed that the variables strongly affected the difference of survivals between both high and low AgNOR groups. This fact suggests that AgNOR score is not an independent prognostic parameter from the already clarified clinicopathologic variables 18-20). In other words, AgNOR score is thought to affect survival and/or prognosis through the difference of clinicopathologic prognostic parameters. In conclusion, AgNOR score was determined in 80 colorectal cancer patients and the relationship between the score and survival was examined. The results suggest that the determination of AgNOR score in the cancer lesions is clinicaliy used as a prognostic parameter but that the score is not an independent prognostic parameter from the already clarified clinicopathologic prognostic parameters. REFERENCES 1. Goessens G. Nucleolar structure. Int Rev Cytol 87: , Franken S, Hernandez YD. The nucleolus and the nucleolar organizer regions (collective review). BioI Cell 56: , Bulletin of Allied Medical Sciences, Kobe

6 AgNOR score in colorectal cancer patients 3. Crocker J, Nar P. Nucleolar organizer regions in lymphomas. J Pathol 151: , Trere D, Pession A, Derenzizi M. The silver-stained proteins of interphasic nucleolar organizer regions asa parameter of cell duplication rate. Exp Cell Res 184: , Derenzini M, Pession A, Trere D. Quantity of nucleolar silver-stained proteins is related to proliferating activity in cancer cells. Lab Invest 63: , Leek RD, Alison MR, Sarraf CEo Variations in the occurrence of silver-staining nucleolar organizer regions (AgNORs) in non-proliferating and proliferating tissues. J Pathol 165: 43-51, Smith R, Crocker J. Evaluation of nucleolar organizer regions-associated proteins in breast malignancy. Histopathol 12: , Crocker J, McGovern J. Nuclelolar organizer regions in normal, cirrhotic and carcinomatous livers. J Clin Pathol 41: , Howat AJ, Giri DD, Wright AL, Underwood JCE. Silver-stained nucleoli and nucleolar organizer region counts are of no prognostic value in thick cutaneus malignant melanoma. J Pathol 156: , Boldy DA, Ayres JG, Crocker J, et al. Inherphase nucleolar organizer regions and survival in squamous cell carcinoma of the bronchus: a 10 year follow-up of 138 cases. Thorax 46: , Rosa J, Metta A, Filipe MI. Nucleolar organizer regions in gastric carcinoma and its precursor stages. Histopathol 16: , Young AN, Wang TY, Liu He. Comparative study of the prognostic value of nucleolar grade and silver-binding nucleolar organizer region in reanl cell carcinomas. J Pathol 166: , Shiro T, Seki T, Naitoh Y, et al. A correlation of argyrophilic nucleolar organizer regions with stages of hepatocellar carcinoma. Cancer 71: 44-49, Toikakanen S, Joensuu H. AgNOR counts have no prognostic value in breast cancer. J Pathol 169: , Aoki K, Sakamoto M, Hirohashi S.,Nucleolar organizer regions in small nodular lesions representing early stages of human hepatocarcinogenesis. Cancer 73: , Miyamura K, Nakamura T, Tabuchi Y, et al. Analysis of argyrophilic nucleolar organizer regions (AgNORs) and DNA ploidy patterns in gastric mp-cancer: correlation with survival. In 1st International gastric cancer congress. Nishi M, Sugano H & Takahashi T (Ed.). Bologna, Monduzzi Editore, pp , Griffinth AP, Butler CW, Roberts P, et al. Silver-stained structures (AgNORs), their dependence on tissue fixation and absence of prognostic relevance in rectal adenocarcinoma. J Pathol 159: , Japanese Research Society for Cancer of the Colon and Rectum. General rules for clinical and pathological studies on cancer of the colon, rectum and anus. Jpn J Surg 13: , Japanese Research Society for Cancer of the Colon and Rectum. General rules for clinical and pathological studies on cancer of the colon, rectum and anus (5th Edition). Tokyo, Kanehara Shuppan, Dukes CEo The classification of cancer of the rectum. J Pathol Bacteriol 35: , 1932 Vol. 12,