Massimo Derenzini

Background: The amount of argyrophilic nucleolar organiser regions (AgNORs) represents a cell kinetics parameter used in tumour pathology for prognostic purposes. AgNOR expression is directly related to the rate of ribosome biogenesis, which has been recently shown to be controlled also by the tumour suppressor proteins pRb and p53. Aims: To ascertain the relative prognostic value of AgNORs and of pRb and p53 expression in breast carcinoma. Methods: This study was carried out on 335 human primary breast carcinomas with a median follow up time of 108 months. AgNORs were measured by morphometric analysis on sections that had been selectively silver stained; expression of p53 and phosphorylated and non-phosphorylated pRb forms was assessed by immunohistochemistry. Results: Patients were divided into groups with low (249) and high (86) AgNOR values; with normal (267) and mutated p53 (68); and with normal (256) and hyperphosphorylated or deleted pRb (79). Univariate analysis of disease free survival showed that AgNORs and the status of pRb and p53 were significantly related to the patients’ clinical outcome. However, among the four groups characterised by different pRb and p53 status, the AgNOR parameter was not capable of distinguishing subgroups of patients with different clinical outcomes. Conclusions: These findings indicate that the prognostic value of the AgNOR parameter depends on the status of the tumour suppressor proteins pRb and p53, and it cannot be ascribed to the relation between AgNORs and the cell proliferation rate.

The present paper discusses the relevance of silver-stained Nucleolar Organizer Regions (AgNOR) to tumour pathology. First, the structural and functional aspects of AgNORs and the biological meaning of their quantitative variations have been reviewed to clearly define which indications can be obtained by the use of AgNOR. In continuously proliferating cells, AgNOR indicates the extent of ribosomal biogenesis, which is strictly related to the rapidity of cell proliferation. Therefore, AgNOR can be considered to represent a marker of cell proliferation rate and, in tumour pathology, should be used only for prognostic purposes. The predictive value of AgNOR, the only parameter which indicates the cell growth rate in situ in routinely processed cyto-histological samples, is strengthened by the combination with markers of cell growth kinetics (e.g. Ki67/MIB1).

The hepatocyte receptor for asialoglycoproteins, which binds and internalizes galactosyl-terminating peptides, was found to be expressed also on the cells of well differentiated hepatocarcinomas. We explored the possibility of obtaining a delivery of antiblastic drugs to hepatocarcinoma cells through this receptor. We conjugated 5-fluoro-2'-deoxyuridine (FUDR) with lactosaminated poly-L-lysine. 5-fluoro-2'-deoxyuridine is an active drug in the treatment of solid tumours, but with toxic effects on intestine and bone marrow. Poly-L-lysine is an galactosyl-terminating carrier which enables preparation of conjugates with very high drug load. We studied the pharmacological activity of poly-L-lysine-5-fluoro-2'-deoxyuridine conjugate on in vitro proliferation of Hep G2 cells, a human hepatocarcinoma cell line. Moreover, we compared the levels of radioactivity in liver, intestine and heart of mice injected with free or conjugated [3H]5-fluoro-2'-deoxyuridine. We found that poly-L-lysine-5-fluoro-2'-deoxyuridine enters into Hep G2 cells through the asialoglycoprotein receptor and, after intracellular penetration, releases the drug in a pharmacologically active form. Administered to mice, the conjugate leads to enhanced accumulation of the drug in liver versus the intestine and the heart. These data support conjugation with poly-L-lysine as a way to obtain drug targeting to those hepatocellular carcinomas which maintain the asialoglycoprotein receptor.

The quantitative distribution of AgNOR proteins has been determined by image cytometry in 52 cervical smears obtained from normal cervix (n = 20), grade I CIN (cervical intraepithelial neoplasia) (n = 3), grade II CIN (n = 5) and grade III CIN (n = 24). No significant difference was demonstrated in the mean AgNOR protein area values between normal cervix, CIN I and CIN II, while AgNOR protein scores of CIN III were significantly greater than those of normal cervix (p < 0.05). AgNOR protein quantity was also determined in 17 colposcopic directed biopsies of patients with CIN lesions (3 CIN II and 14 CIN III) already studied by cytological analysis. When AgNOR protein values of histological sections and corresponding cytological smears were compared by linear regression analysis, a significant correlation was found (r = 0.74, p < 0.05). Our results demonstrate that AgNOR protein quantity of cervical smears actually reflects that of the cervical epithelium in situ and may therefore be useful for the cytological diagnosis of cervical lesions.

Conventional histological examination of echo-guided biopsy specimens can be inconclusive in small nodular lesions in cirrhotic livers. We investigated the diagnostic potential of cytochemical analysis of dipeptidyl-peptidase IV (DPP IV), of image analysis of nuclear DNA content, and of interphase silver-stained nucleolar organizer regions (AgNORs) in 12 cases of small (13- to 29-mm in diameter) hepatic nodules visualized in cirrhotic patients by ultrasonography. All cases underwent an echo-guided liver biopsy at the time of detection and in none of them were histological signs of malignancy found. Characterization with the above-mentioned techniques was always done at the time of histological examination. These patients underwent a mean (±sd) follow-up of 27.0 (±11.2) months after biopsy, with repeated ultrasound (US) examinations. In the seven patients with subsequent neoplastic growth, DPP IV score was altered in five of six; the fraction of mononucleated polyploid cells was altered in six of seven; and the AgNOR quantity exceeded the cutoff value of 4 μm² in five of five cases. Among the five lesions whose US appearance remained unchanged during the follow-up, only one abnormality (AgNORs) was found in one case. The combined cytochemical analysis of DPP IV, nuclear DNA content, and quantitative evaluation of interphase AgNORs in biopsy samples may contribute to the differential diagnosis of hepatocellular nodules of uncertain type in the cirrhotic liver.