Demonstration of nucleolar organizer regions in intrahepatic bile duct carcinoma by the silver-staining technique

Abstract— A silver colloid technique to identify argyrophilic nucleolar organizer region associated protein (AgNOR) was applied to 43 cases of intrahepatic bile duct carcinoma (cholangiocarcinoma, CC), 2 with bile duct adenoma (BDA), 5 with focal duct epithelial hyperplasia (FEH) associated with hepatolithiasis, 15 with posthepatitic ductular proliferation (PHDP) associated with massive or submassive hepatic necrosis and 20 of normal liver. In the present study, only discrete, easily counted black dots within nuclei and silver-stained nucleolus were counted under a magnification of X 400 without oil-immersion objectives. The mean AgNOR count of CC was significantly higher than those of BDA, FEH, PHDP and normal controls (P>0.05, P>0.001, P>0.01, and P>0.001, respectively). Among CCs the mean AgNOR numbers of papillary adenocarcinoma (pap), moderately (tub2) and poorly differentiated (por) adenocarcinoma, and adeno-squamous carcinoma (as) were significantly higher than that of normal controls (P > 0.01, P > 0.001, P > 0.001 and P > 0.001, respectively), and those of tub2, por and as were also significantly higher than those of BDA, FEH and PHDP, whereas that of well differentiated tubular adenocarcinoma (tub 1) was not different from those of BDA, FEH, PHDP and normal controls, and that of pap was not different from those of BDA, FEH and PHDP. The mean numbers of AgNORs of BDA and FEH were not different from that of normal controls, whereas that of PHDP was significantly higher than that of normal controls (P > 0.01). Interestingly, the mean AgNOR counts of tubular adenocarcinoma were increased with histologic tumor grades. All cases with AgNOR counts of more than 2.24 had CC. Furthermore, not only quantitative but also striking qualitative abnormalities of AgNORs were seen in CCs. Large and/or irregularly shaped AgNORs without uniformity in size and density were characteristic for CCs, even in well differentiated adenocarcinoma, in contrast to the small, round, regular AgNORs seen in normal bile duct epithelium and rather large but regular AgNORs seen in benign bile duct lesions. These results indicate that the enumeration of AgNORs in various bile duct lesions is useful to identify moderate and high grade CCs and to evaluate tumor grade, but not useful to discriminate well differentiated adenocarcinoma from benign lesions. In case of normal or equivocal AgNOR counts, the presence of large and/or irregularly shaped AgNORs without uniformity in size and density would favor a diagnosis of carcinoma rather than benign counterparts.