Cell Kinetic Study of Human Carcinomas Using Bromodeoxyuridine

Abstract. Cell kinetics in human malignant tumours were studied in vivo using bromodeoxyuridine (BrdU) and immunohistochemistry. BrdU was administered to twenty-four patients with gastric cancer at a dose of 1 g pre-operatively. Specimens were obtained during the operation, fixed in 70% ethanol and embedded in paraffin. BrdU-incorporating cells were detected by immunohistochemical staining using anti-BrdU monoclonal antibody. the labelling index (LI), determined by counting tumour cells microscopically, ranged from 4.0 to 41.4%. the LI was higher at the site of invasion than in the central area of the tumour, but no correlation was found between histological differentiation and LI. the LI of stage I gastric cancer was statistically lower than that of stage II, III and IV gastric cancers (P < 0.005). This technique, which is less cumbersome and time-consuming than using radioactive isotopes of thymidine, appears to be useful for studying cell kinetics of human malignant tumours.     

Cell kinetic study of human carcinomas using bromodeoxyuridine

Cell kinetics in human malignant tumours were studied in vivo using bromodeoxyuridine (BrdU) and immunohistochemistry. BrdU was administered to twenty-four patients with gastric cancer at a dose of 1 g pre-operatively. Specimens were obtained during the operation, fixed in 70% ethanol and embedded in paraffin. BrdU-incorporating cells were detected by immunohistochemical staining using anti-BrdU monoclonal antibody. The labelling index (LI), determined by counting tumour cells microscopically, ranged from 4.0 to 41.4%. The LI was higher at the site of invasion than in the central area of the tumour, but no correlation was found between histological differentiation and LI. The LI of stage I gastric cancer was statistically lower than that of stage II, III and IV gastric cancers (P less than 0.005). This technique, which is less cumbersome and time-consuming than using radioactive isotopes of thymidine, appears to be useful for studying cell kinetics of human malignant tumours.     

Proliferative activity of gastric epithelial cells in Helicobacter pylori infected children

Helicobacter pylori infection has been associated with gastric carcinogenesis. Gastric epithelial cells proliferative rate is accelerated in H. pylori infected adult patients. Our study was performed to evaluate proliferative cell activity in gastric epithelium in the course of H. pylori infection in the early stage of its natural history. Gastric antral biopsy specimens were obtained from thirteen H. pylori positive and seven negative children. To assess replication rates we used nucleolar organiser regions staining with colloidal silver nitrate technique (AgNOR). The number of AgNORs per nucleus, area of single AgNOR, and the quotient of these two parameters (AgNOR content) were analysed. The mean area of AgNOR was lower in H. pylori positive than in negative children. Conversely, both the mean number of AgNOR per nucleus and AgNOR content were higher in infected than non infected subjects. These results show accelerated proliferation of gastric antral epithelial cells in the course of H. pylori infection in children. Such alteration of cell replication occurring in an initial phase of natural history of long lasting infection provides an explanation for the association between acquisition of H. pylori infection in the first years of life and the development of gastric cancer.     

Manual and automated AgNOR count in differentiating reactive mesothelial from metastatic malignant cells in serous effusions

OBJECTIVE: To distinguish reactive mesothelial cells from malignant cells in serous effusions using manual and automated methods of enumeration of argyrophilic nucleolar organizer regions (AgNORs). STUDY DESIGN: In this prospective study, 38 samples of benign (19 cases) and malignant (19 cases) serous effusions were included. AgNOR stain was used in each case along with routine Papanicolaou stain. The smears were examined under an oil immersion objective, and AgNOR dots were counted by direct observation independently by 2 observers. Automated AgNOR counting and morphometry were performed with a Quantimet 600 image cytometer (Leica, Cambridge, England). At least 100 cells were counted in each case. The number of AgNOR dots in individual cells, AgNOR area, nuclear area, AgNOR vs. nuclear area and nuclear perimeter were measured. Data on benign and malignant cells were compared. RESULTS: The AgNOR dots were discrete and smaller in benign effusion cases as compared to coarse and aggregated in malignant effusion cases. In benign reactive effusion cases the mean number of AgNOR dots per nucleus was 2.33 +/- 0.71 and 2.83 +/- 1.15 by the manual and automated method, respectively, whereas that for malignant effusion cases was 7.48 +/- 2.51 and 8.09 +/- 1.69 by the manual and automated method, respectively. Mean total AgNOR areas in benign and malignant groups were 4.77 +/- 2.66 microns 2 and 38.22 +/- 13.71 microns 2, respectively. Mean nuclear area, nuclear perimeter and ratio of AgNORs vs. nuclear area were 48.72 +/- 19.30 microns 2, 24.68 +/- 10.25 microns and .098 in benign effusion cases as compared to 174.25 +/- 82.36 microns 2, 69.03 +/- 27.23 microns and 0.22 in malignant effusion samples. All these values were significantly higher (P < .001, Student's t test) in malignant cells as compared to benign reactive cells. CONCLUSION: AgNOR dot enumeration, AgNOR area and ratio of AgNORs to nuclear area are valuable adjuncts to cytomorphology in differentiating reactive mesothelial cells from malignant cells in serous effusions. Automated AgNOR counting is rapid and less cumbersome.     

Morphometry and digital AgNOR analysis in cytological imprints of benign, borderline and malignant serous ovarian tumours

AIM: The aim of the study was to determine values of a quantitative morphometry analysis of nuclear characteristics and argyrophilic nucleolar organizer regions (AgNORs) in differential cytodiagnosis of benign, atypically proliferating (borderline) and malignant serous ovarian tumours. METHODS: Cytological imprints of benign (n = 20), borderline (n = 19) and malignant (n = 20) ovarian serous tumours were analysed. A computerized, digital analysis was used to determine morphometric nuclear features, the number and characteristics of single AgNORs, cluster AgNORs, total AgNOR and AgNOR area/nucleus (relative area) ratio. According to their size AgNORs were classified in three categories. A one-way variance analysis and post hoc test (Scheffé) were used for statistical analysis. RESULTS: The morphometric nuclear analysis showed that benign, borderline and malignant serous ovarian tumours are statistically different (P < 0.001) according to the area and outline, the values being highest in malignant tumours and lowest in the borderline group. Digital analysis of AgNORs in benign, borderline and malignant groups showed that the total AgNOR number increases with progression of the lesion (meaning tumour malignancy) significantly (P < 0.001) between benign and malignant as well as between borderline and malignant serous ovarian tumours (P < 0.001). The progression of the lesion malignancy was accompanied by a significant (P < 0.001) progressive increase of the total and relative AgNOR area per nucleus. The AgNOR size increases from benign to malignant tumours and a statistically significant difference (P < 0.001) was observed in all three groups regarding small and large AgNORs. CONCLUSION: Combining different markers of morphometric nuclear characteristics and AgNOR values could improve differential cytodiagnosis of benign, borderline and malignant serous ovarian tumours.     

Prognostic significance of standardized AgNOR analysis and Ki-67 immunostaining in gastrointestinal stromal tumors

OBJECTIVE: To evaluate the prognostic utility of argyrophilic nucleolar organizer region (AgNOR) protein parameters and Ki-67-immunostained growth fraction (Ki-67 labelling index) and to correlate AgNORs with Ki-67 LI and the main clinicopathologic parameters in gastrointestinal stromal tumors (GISTs). STUDY DESIGN: On 55 patients with surgically excised GISTs, visualization and quantification of AgNORs were performed as specified in the guidelines of the Committee on AgNOR Quantification. RESULTS: AgNOR protein area (NORA) > or = 5.28 microns 2 was statistically associated with mitotic rate > or = 5 x 10 high-power fields (hpfs) (P < .001) and presence of necrosis (P < .001); Ki-67 LI > or = 9.69% was significantly associated with mitotic rate > or = 5 x 10 hpfs (P < .001), size > or = 5 cm (P = .033) and presence of necrosis (P < .001). Ki-67 LI and NORA strongly correlated. Preliminary Kaplan-Meier survival analysis showed that an increased value of NORA, Ki-67 LI, mitotic rate, tumor size and presence of necrosis had a negative influence on patient survival. Multivariate regression analysis showed that only NORA and Ki-67 LI were independent parameters in predicting the clinical outcome for patients with GISTs. Mitotic rate and necrosis remained as independent prognostic factors when NORA and Ki-67 LI were not allowed to enter in models. CONCLUSION: AgNOR protein quantity, as determined by image cytometry, and Ki-67 immunostaining seem to represent reliable predictive parameters in GISTs and are independent of mitotic rate, tumor dimension and necrosis.     

AgNOR count in exfoliative cytology of normal buccal mucosa. Effect of smoking

OBJECTIVE: To compare the argyrophilic nucleolar organizer region (AgNOR) count of cells collected from normal buccal mucosa of cigarette smokers with that obtained from nonsmokers. STUDY DESIGN: Cytologic smears of normal buccal mucosa from 20 smokers and 20 nonsmokers were stained for AgNORs. The AgNOR count was established on 100 cells. The count values of groups were compared and analyzed using Student's unpaired t test. RESULTS: The AgNORs were round and had a clustered distribution in both groups. The mean AgNOR count was statistically higher in cells of smokers than nonsmokers (P < .01). CONCLUSION: Analysis of AgNORs suggests that cigarette smoking influences proliferative activity in cells of normal buccal mucosa.     

Morphometric analysis of AgNORs in nonkeratinizing carcinoma and adjacent normal epithelia in the nasopharynx

OBJECTIVE: To evaluate the proliferative activity of different types of nonkeratinizing carcinoma and adjacent normal epithelia in the nasopharynx by the quantitative assessment of argyrophilic nucleolar organizer region (AgNOR) proteins. STUDY DESIGN: Silver staining of nucleolar organizer regions (NORs) was applied to 70 paraffin sections of nonkeratinizing carcinoma in nasopharyngeal biopsies. Fifty-four of the 70 cases had differentiated nonkeratinizing carcinoma (DNC), and the remaining 16 had undifferentiated carcinoma (UC). Nineteen of these 70 samples proved to contain, besides carcinoma, normal epithelia (NE), which was used as a control. The epithelial cells and cancer cells were analyzed for their AgNOR features by image cytometric analysis. RESULTS: As compared with normal epithelia, significant differences were found in mean nuclear area, AgNOR count, mean AgNOR area, AgNOR area ratio and AgNOR area/count ratio between NE and DNC (P < .05) and in mean nuclear area, mean AgNOR area and AgNOR area/count ratio between NE and UC (P < .001). Further, the differences in mean nuclear area, mean AgNOR area and AgNOR area/count ratio were statistically significant between DNC and UC. CONCLUSION: The evaluation of AgNORs is a useful histologic assessment of rapidity of cell proliferation in malignant and benign lesions and demonstrated that UC had more rapidly proliferative activity than DNC in this study.     

鼻咽癌及癌旁上皮细胞的AgNORs定量分析及其生物学意义

目的通过鼻咽癌及癌旁上皮的细胞核仁组成区蛋白(NORs)图像定量分析,评价其反映细胞群体增生能力及分化程度等生物学意义。方法对70例鼻咽癌石蜡切片进行银染(AgNORs),应用CAS200图像分析仪分别测定29例癌旁正常腺上皮、19例增生／异型增生柱状上皮、10例增生／异型增生鳞状上皮、54例分化性非角化性癌和16例未分化癌的细胞群体AgNORs参数值并作比较分析;结果每核AgNOR计数、每核AgNOR面积和平均AgNOR面积／粒从正常腺上皮至增生／异型增生柱状上皮至分化性非角化性癌或未分化癌呈现显增加,平均核面积、每核AgNOR面积和平均AgNOR面积／粒从正常腺上皮至增生／异型增生鳞状上皮至未分化癌呈现显增加,差异均有显性,但增生／异型增生鳞状上皮与分化性非角化性癌的AgNOR数值差异没有显差异;与分化性非角化性癌比较,未分化癌的平均核面积、每核AgNOR面积和平均AgNOR面积／粒显增加。结论．AgNORs形态定量分析反映了鼻咽癌及癌旁不同类型鼻咽上皮的细胞增生活性和组织细胞分化程度,但不能反映细胞是否恶性转化;不同组织类型鼻咽癌细胞存在增殖能力的差异,其增生差异程度可能是影响病人对放射线治疗敏感性及其预后的重要因素之一。     

Digital image analysis of silver-stained nucleolar organizer region--associated proteins in endometrial cytologic samples

OBJECTIVE: Digital image analysis was applied to determine the number, area and size of silver-stained nucleolar organizer regions (AgNORs) in cytologic samples from curettage in normal, hyperplastic and malignant endometrium. STUDY DESIGN: Thirty-two archival cytologic smears from curettage (previously stained by the Papanicolaou method) with the histologic diagnosis (4 inactive endometrium, 5 secretion, 5 proliferation, 5 simple hyperplasia, 5 complex hyperplasia, 3 atypical hyperplasia, 5 adenocarcinoma, grade 1) were analyzed with the AgNOR technique. Count, area and size of AgNORs were analyzed in 50 cells per sample using a magnification of 1,000x. Quantitative analysis was performed on an SFORM digital imaging system. Data were analyzed with the SPSS/PC+ program. Mann-Whitney and chi 2 tests were performed. RESULTS: The average value of AgNOR count increased from normal to hyperplastic endometrium and well-differentiated adenocarcinoma. Differences were significant except between atypical hyperplasia and adenocarcinoma. Four, five and more AgNORs in 40% or more of the nuclei were found in complex and atypical hyperplasia and adenocarcinoma. Proliferation, and simple and atypical hyperplasia had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic and well-differentiated adenocarcinoma. Differences were statistically significant. AgNOR size in well-differentiated adenocarcinoma was significantly different from that in normal endometrium and different grades of hyperplasia. CONCLUSION: Digital image analysis of AgNOR count, area and size enabled a distinction to be made between normal, hyperplastic and malignant endometrium.     

Expression of argyrophilic nucleolar organizer regions (AgNORs) can be used to assess cellular proliferation as shown in rat thymic sections

Summary Silver-stained nucleolar organizer regions (AgNORs) were studied in thymic sections from 4- and 30-day-old rats. By direct examination under the light microscope cells with a low or high content of AgNORs (type I and type II cells, respectively) were identified and their relative numbers calculated. The mean area of AgNORs per cell was calculated for each type of cell and age group. Additionally, the proportion of cells labelled with bromodeoxyuridine was calculated in sections from the same animals. Visual identification of type I and type II cells was confirmed by a significant (p < 0.01) difference in the mean AgNOR area in both types of cell. Both the proportion of cells with a high expression of AgNORs (type II cells) and that of bromodeoxyuridine-labelled cells were significantly greater (p < 0.01) in 4-day-old rats than in 30-day-old rats. A significant correlation was found between both variables (R² = 0.45;p = 0.002), the relation being best between both variables and age (R² = 0.91;p = < 0.001). These data offer support for an easy interpretation of the AgNOR reaction in which the proportion of cells with a high expression of AgNORs can be used as an index of proliferative activity in a tissue sample.     

Nucleolar organizer regions in aspirates of malignant lymphomas and benign disorders of the lymph nodes.

Silver staining of nucleolar organizer regions (AgNOR) was used to differentiate malignant lymphoma and chronic lymphadenitis. Aspiration smear samples from lymph nodes of 120 cases, including 43 non-Hodgkin's lymphoma, 3 Hodgkin's disease, 56 chronic lymphadenitis, 7 tuberculosis, 6 reactive hyperplasia and 5 samples from other diseases (epidermoid cyst, branchial cyst, mixed tumor, lymphoepithelioma and nodulous disease), were investigated. The number of AgNORs in 200 cells in each sample was counted, and the mean +/- SD in each disease was calculated: non-Hodgkin's lymphoma, 6.58 +/- 2.37; Hodgkin's disease, 4.22 +/- 0.5; chronic lymphadenitis, 1.16 +/- 0.1; tuberculosis, 1.13 +/- 0.14; reactive hyperplasia, 1.48 +/- 0.25; other diseases, 1.47 +/- 0.31. The results indicate that the AgNOR count in malignant lymphoma differed highly significantly from that in benign disease (P less than .001). The size of AgNORs in malignant lymphoma and chronic lymphadenitis was measured, and the maximum diameter and area of lymphocyte and lymphoma cell were: lymphocyte, 0.93 +/- 0.12 microns, 0.61 +/- 0.13 microns 2; lymphoma cell, 0.83 +/- 0.22 microns, 0.50 +/- 0.25 microns 2. The AgNOR sizes in malignant lymphoma were significantly smaller than in chronic lymphadenitis (P less than .001).     

Different proliferation patterns in breast cancer: AgNOR measurements in ER-negative and ER-positive tumor cells.

The relation between estrogen receptors (ER) and argyrophilic nucleolar organizer regions (AgNORs) in situ within human breast cancer cells was analyzed. For AgNOR measurements in 49 invasive breast carcinomas, a new reproducible staining method for dual demonstration of ER and AgNORs was applied. Quantitative AgNOR variables were determined in ER-positive and ER-negative tumor cells by digital image analysis. The relationships between AgNOR parameters of ER-positive and ER-negative cells and other prognostic factors of breast cancer [Bloom-Richardson-Grading and growth fraction (Ki-67 index)] were investigated. A higher AgNOR content in ER-negative cells and a special clustering phenomenon in ER-positive tumor cells were found. Correlation with other criteria of malignant potential could be exclusively demonstrated for ER-negative cells. ER-negative cells of breast cancer can be characterized as the more malignant and possibly prognosis-dictating cell fraction. Thus, ER-negative cells probably contribute more to the progression of the tumor disease and furthermore to the prognosis than ER-positive cells. We recommend measurement AgNORs exclusively in ER-negative cells of breast cancer.     

Morphometric analysis of AgNORs in thin-layer, liquid-based liver specimens

OBJECTIVE: To detect argyrophilic nucleolar organizer regions (AgNORs) in ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A.) liver fine needle aspiration (FNA) specimens and to define the diagnostic value of their quantitative analysis in the evaluation of hepatic lesions. STUDY DESIGN: ThinPrep liquid-based FNA biopsy specimens from 49 malignant and benign liver lesions were resampled, fixed in 95% ethanol and stained with the AgNOR technique in accordance with the 1-step colloid method. The specimens included 11 benign and 38 malignant lesions (23 poorly differentiated hepatocellular carcinomas [HCCs] and 15 poorly differentiated metastatic adenocarcinomas [MCs]). Morphometric analysis was performed using a Zeiss Axiolab microscope (Carl Zeiss GmbH, Jena, Germany) with a mechanical stage fitted with a Sony-iris CCD videocamera (Tokyo, Japan). The videocamera was connected to a Pentium III P/C (Intel Corp., Santa Clara, California, U.S.A.) loaded with the appropriate image analysis software. The measurements were performed with ImageScan software (Jandel Scientific, Erkrath, Germany). The number of AgNORs per nucleus (NN) and the total area per nucleus occupied by AgNORs (AR) were calculated semiautomatically. Statistical analysis was performed using the SPSS software package (Chicago, Illinois, U.S.A.). RESULTS: The least significant deviance test for multiple comparisons revealed that NN differed significantly between the 3 groups of samples examined (P < .0001). The mean NN values in HCCs and MCs were significantly different (P < .0001). Logistic regression model demonstrated that as NN increased, the probability of a MC diagnosis decreased (<4%). AR values were different at a statistically significant level only between benign and malignant specimens (P = .00006), not between HCCs and MCs (P = .933). CONCLUSION: Quantitative analysis of AgNORs in ThinPrep specimens could be a diagnostically useful method in liver disease.     

Silver-stained nucleolar organizer proteins in chondrosarcoma.

Silver-stained nucleolar proteins (AgNORs) were counted in primary chondrosarcomas of three histologic grades and in metastatic chondrosarcomatous lesions in the lung. The AgNOR numbers of neoplastic cells in primary tumors increased stepwise from grade 1 (4.42 +/- 1.11) through grade 2 (4.94 +/- 1.31) to grade 3 (6.97 +/- 1.10). There was a significant difference in AgNOR numbers between grade 3 and both grades 1 and 2 (p less than 0.001). Furthermore, the mean number of AgNORs in metastatic lesions (9.75 +/- 0.83) was significantly higher than that in primary sites (p less than 0.001). The number of AgNORs therefore reflects the grade of the chondrosarcoma. The results in the present study indicate that silver colloid staining is a useful technique for determining the histologic grade and evaluating the proliferative activity of chondrosarcomas.     

Nuclear morphometry and AgNOR quantification: computerized image analysis on ovarian mucinous tumor imprints

OBJECTIVE: To determine the morphometric characteristics of nuclei and silver-stained nucleolar organizer regions (AgNORs) on cytologic imprints and their value in differential cytodiagnosis of benign, atypical proliferative (borderline) and malignant ovarian mucinous tumors. STUDY DESIGN: Forty-six mucinous ovarian tumor imprints (16 benign, 15 borderline, 15 malignant), were analyzed. Nuclear area, outline, "shape factor" and "form factor" were measured on Papanicolaou-stained smears. AgNOR quantification included 7 variables related to the number and area of single, cluster, total and relative AgNOR content per nucleus and the size distribution of AgNORs. RESULTS: Nuclear area and shape factor allowed distinguishing borderline and malignant tumors. The nuclear area in benign tumors was larger than that in borderline tumors; malignant tumors had the highest values. Single and cluster AgNORs were statistically significantly different in borderline tumors compared with malignant tumors, except for the cluster AgNOR area. The total AgNOR area, number and relative area increased from benign through malignant tumors, with statistically significant differences among all groups. By AgNOR size distribution, small AgNORs discriminate malignant from borderline and benign tumors. CONCLUSION: Combining nuclear morphometry and AgNOR analysis on cytologic imprints could be a diagnostically useful method in the assessment of mucinous ovarian tumors.     

Ki-67 and AgNOR proliferative markers as diagnostic adjuncts to fine needle aspiration cytology of thyroid follicular lesions

OBJECTIVE: To differentiate hyperplastic nodules (HPN), follicular adenoma (FA) and follicular carcinoma (FCA) of the thyroid by cytomorphologic features combined with argyrophilic nucleolar organizer regions (AgNORs) and Ki-67 proliferative markers on fine needle aspiration cytology. STUDY DESIGN: Cytomorphologic patterns, along with two proliferation markers, Ki-67 and AgNORs, in fine needle aspirates of 123 histologically confirmed cases of thyroid follicular lesions, including 39 hyperplastic nodules, 70 follicular adenomas and 14 cases of follicular carcinomas, were recorded. RESULTS: Mean AgNOR (mAgNOR) counts and Ki-67 labelling index (LI) were consistently higher in FCA in comparison to FA and HPN irrespective of the cytologic patterns in fine needle aspiration smears. Between benign and malignant lesions, an overlap of 1.83% at the cutoff point of 4.0 was observed in cases of mAgNORs, whereas it was 11.09% at a cutoff of 5.0 in cases of Ki-67 LI. CONCLUSION: mAgNOR counting in fine needle aspiration smears is more sensitive, simple and cost effective as compared to Ki-67 LI for differentiating between benign and malignant thyroid follicular neoplasms.     

AgNOR numbers in rat pituitary corticotrophs following adrenalectomy or corticotrophin releasing factor administration

Using a combined silver staining/immunoalkaline phosphatase technique, nucleolar organiser regions (AgNORs) were visualised and quantified in rat anterior and intermediate lobe pituitary corticotrophs following bilateral adrenalectomy or sham surgery. Compared to sham operated animals, the mean number of AgNORs was increased in anterior lobe corticotrophs in adrenalectomized rats and there was a shift to the right in the distribution. At 2 weeks after adrenalectomy, AgNOR numbers were greater than at 6 weeks. AgNOR numbers were also quantified in anterior lobe corticotrophs of intact rats receiving daily intraperitoneal injections of ovine CRF-41 at 50 micrograms/kg, which has been shown to stimulate ACTH release and to produce morphological evidence of increased corticotroph stimulation. CRF-41 did not produce an increase in AgNOR numbers, compared to saline injected controls.     

Prognostic value of nucleolar organizer regions in neuroendocrine tumours of the lung

The value of the number and size of the nucleolar organizer regions (NORs) as prognostic indicators in human neuroendocrine lung tumours was evaluated in a quantitative study of 57 cases, including 33 small cell carcinomas (SCLCs), 9 well-differentiated neuroendocrine carcinomas (WDNECs) and 15 classic carcinoids. NORs were visualized on paraffin sections by an argyrophilic technique (AgNOR) and measured by automatic image analysis. In each case, the mean number and area of AgNORs were evaluated; the results were compared with clinical follow-up and survival. AgNOR values for both number and area were significantly higher in SCLCs than in WDNECs and carcinoids. WDNECs had insignificantly higher AgNOR values than carcinoids. Among SCLCs, AgNOR values of the oat cell subtype and the intermediate cell subtype did not differ significantly. Regardless of the histological tumour type, AgNOR values strongly correlated with prognosis, with more and larger AgNORs indicating a more progressive clinical course. In the present study we demonstrate for the first time that the biological behaviour of neuroendocrine lung tumours is correlated with the number and size of AgNORs. Thus the measurement of AgNORs may serve as an additional prognostic indicator in these neoplasms, particularly in the separation of SCLCs from WDNECs with a more favourable prognosis.