Assessment of proliferative activity of thyroid Hürthle cell tumors using PCNA, Ki-67 and AgNOR methods

We have undertaken an attempt to compare the application efficacy of the proliferative activity markers in differential diagnosis of thyroid Hürthle cell tumors (HCT) using the PCNA and Ki-67 labeling and AgNOR visualisation techniques. The present work is a retrospective analysis of 78 Hürthle cell tumors: 20 Hürthle cell carcinomas (HCC), 32 Hürthle cell adenomas (HCA) and 26 hyperplastic nodules with Hurthle cell metaplasia (HCM). Five microm sections were stained according to AgNOR technique and labeled with antibodies against PCNA and Ki-67. AgNOR dot count in the nucleus and proliferative index (PI - percentage of cells expressing PCNA and Ki-67) in randomly chosen nuclei (100 in case of AgNOR and over 1000 in case of PI) were evaluated in each slide. The mean values of AgNOR dot count, PI-PCNA and PI-Ki-67 in HCC, HCA and HCM were respectively: 5.1, 61.3 and 54.9; 3.4, 42.4 and 38.6 and 2.5, 39.3 and 34.3. Statistically significant difference was found in all the proliferative activity markers between malignant and benign tumors: HCC:HCA (p<0.01) and HCC:HCM (p<0.001). There was no statistically significant difference between HCA and HCM.     

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.     

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。     

Quantitative AgNOR assessment of cancer cell populations in undifferentiated nasopharyngeal carcinoma

OBJECTIVE: To evaluate the proliferative activity of cancer cell populations in undifferentiated nasopharyngeal carcinoma (NPC) by quantitative assessment of argyrophilic nucleolar organizer regions (AgNOR) proteins. STUDY DESIGN: Silver-staining of NORs was applied to 18 paraffin sections of undifferentiated NPC containing a vesicular nuclear cancer cell population (VNCC) and spindle-shaped cancer cell population (SSCC). Various features of AgNORs in these two cell populations were analyzed by image cytometry and compared. RESULTS: Mean AgNOR count, mean AgNOR area and AgNOR area-count ratio of VNCC were statistically significantly higher than those of SSCC, while no significant difference was found in AgNOR area/nuclear area ratio between the two cancer cell populations. CONCLUSION: The different cancer cell populations with their distinct morphologic characteristics in undifferentiated NPC have different proliferative activity. The relationship between proliferative heterogeneity of cancer cell populations in undifferentiated NPC and its sensitivity to radiotherapy awaits further study.     

Evaluating Mitotic Activity in Canine and Feline Solid Tumors: Standardizing the Parameter

Three different methods for evaluating mitotic activity (mitotic count, mitoses/area, mitotic index) were applied to different types of canine and feline solid tumors to determine the method that is most objective and correlates best with other parameters of cell proliferation. Mitotic activity was evaluated on toluidine blue stained his-tological sections. Slides stained with histochemical (AgNOR proteins) and im-munohistochemical (MEB1, PCNA) markers of cell proliferation were available for each case. Quantitation of mitotic activity and cell proliferation parameters was performed with an image analyzer. Mitotic activity assessment was compared with cell proliferation indices and its ability to discriminate tumors grouped on histologically based criteria including the histological type, malignant or benign characteristics, and grade. A significant correlation by linear regression analysis with other parameters assessing cell proliferation revealed that mitotic index correlated 100% and mitoses/area and mitotic count correlated 40% of the time. In discriminating the proliferative activity of tumors grouped by histological criteria, mitotic index and mitotic count revealed 100% concordance with the other parameters of cell proliferation, while mitoses/areas showed 80% concordance.     

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.     

Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions

Nucleolar organizer regions are nucleolar components that contain proteins that are stained selectively by silver methods; they can be identified as black dots throughout the nucleolus and are known as silver binding nucleolar organizer regions (AgNOR). The number of AgNOR is related to the cell cycle and the proliferative activity of the cells. We investigated AgNOR using exfoliative cytology smears of potentially malignant oral lesions. Eighty individuals were divided into four equal groups: healthy controls, oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. The mean number of AgNOR in each study group gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. The proliferative index was increased in the oral premalignant and malignant patients compared to normal subjects. The mean AgNOR size gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. Spherical shaped AgNOR were most common in controls, whereas large, clustered and kidney shapes were most common in oral squamous cell carcinoma. Multiparameter analysis of AgNOR in oral exfoliative smears is a simple, sensitive and cost-effective method for differentiating premalignant from malignant lesions and can be used in conjunction with routine cytomorphological evaluation.     

AgNOR count in resting cells (resting NOR) is a new prognostic marker in invasive bladder tumor.

PURPOSE: We have previously demonstrated that the AgNOR count in proliferating cells is a predictor of tumor recurrence in superficial bladder tumor (J. Urol. 162 (1999), 63-68). In the present study, we evaluate the type of AgNOR associated with cell cycles as a prognostic factor in invasive bladder tumor using a double staining technique employing both AgNOR and MIB-1 labelling. MATERIALS AND METHODS: Forty-four paraffin sections of invasive bladder tumors were stained simultaneously with AgNOR and MIB-1. The number of AgNORs in proliferating (MIB-1 positive) or resting (MIB-1 negative) cells were counted from a total of 100 nuclei. Correlations between MIB-1 associated AgNOR count and clinicopathological parameters were statistically analyzed. RESULTS: The AgNOR count in proliferating cells (proliferating NOR) was significantly higher than that in resting cells (resting NOR) (p<0.01). The resting NOR in tumors with distant metastases was significantly higher than that in tumors without metastases (p<0.05). Patients with a low resting NOR tumor had a better prognosis than those with a high resting NOR tumor, whereas the proliferating NOR was not associated with survival. Survival analysis revealed that the resting NOR was the most powerful prognostic marker in patients with invasive bladder tumor (p<0.05). CONCLUSIONS: Resting NOR had a predictive value in the prognosis of patients with invasive bladder tumor.     

Progression curves for endometrial lesions

OBJECTIVE: To derive a numeric measure for the progression of endometrial lesions as a baseline study for an eventual assessment of chemopreventive intervention efficacy. STUDY DESIGN: Tissue sections from normal endometrium at the proliferative and secretory phase, simple hyperplasia, atypical hyperplasia from cases free of concomitant adenocarcinoma and adenocarcinoma of the endometrium were recorded at high spatial resolution. Six cases from each diagnostic category were chosen as "typical," and 60 epithelial nuclei were randomly selected for measurement for each case. Discriminant analyses were carried out to derive a direction of progressive change in feature space and to correct the progression curve for the presence of cells not expressing progressive change among the random sample of nuclei. RESULTS: A well-conditioned progression curve was derived based on the mean discriminant function scores for each diagnostic category and the mean nuclear abnormality of the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. The lesion signatures showed a clear trend toward extension into the range of higher nuclear abnormalities with increasing progression. There was an indication that abnormal endometrial lesions may comprise cases with distinctly different degrees of nuclear abnormality. CONCLUSION: A numeric assessment of lesion progression for endometrial lesions, based on karyometric measurements, is possible. The data suggest that additional analysis may provide further characterizing information for individual lesions.     

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.     

Cell proliferation patterns in canine infundibular keratinizing acanthoma and well differentiated squamous cell carcinoma of the skin

The aim of the present study was to investigate if the evaluation of cell proliferation of the well differentiated squamous cell carcinoma (WDSCC) and infundibular keratinizing acanthoma (IKA) could be useful in the differential diagnosis between these two tumours. Eighteen IKAs and ten WDSCCs were selected for this study. Two different methods were used to assess the activity of cell proliferation: MIB1 immunohistochemical detection and AgNOR proteins silver staining. The quantification of proliferative parameters was performed by means of an image analyzer and expressed as MIB1 index and AgNOR area (MNORA). Both MIBI immunohistochemical and AgNOR histochemical patterns were different in WDSCC and IKA; moreover analysis of variance showed a significant difference for both parameters employed (MIB1 index, MNORA) between WDSCC and IKA (P<0.003 for MIB1 index; P<0.0001 for AgNOR area). The results show that canine WDSCC and IKA have a different proliferative behaviour and the assessment of cell proliferation can be considered as a useful adjunctive tool to the histopathological investigation in the differential diagnosis of these tumours.     

Proliferative activity of the thyroid oxyphilic tumor cells estimated by means of quantitative analysis of silver stained nucleolar organizer regions (AgNORs)

Promising results of studies on different neoplasms, by means of morphometric analysis of argyrophilic nucleolar organizer regions (AgNORs), known as proliferative activity marker, made us undertake an attempt to evaluate of proliferative activity in Hürthle Cell Tumors using the same technique. 78 cases including 20 Hürthle Cell Carcinomas (HCC), 32 Hürthle Cell Adenomas (HCA) and 26 hyperplastic nodules with Hürthle Cell Metaplasia (HCM), diagnosed in the Department of Clinical Pathology, Medical Academy of Bialystok in the years 1990-2000, were subjected to analysis. For visualization of the NORs we used the D. Ploton et al. technique. Mean AgNOR count and NORDS (NOR distribution score--the percentage of nuclei with at least 5 argyrophilic granules) were estimated in each case in 100 randomly chosen nuclei. Non-parametric Mann-Whitney U-test was applied to determine statistically significant differences. RESULTS: Mean AgNoRs counts and NORDS values were 5.1 and 52%, 3.4 and 26%, 2.5 and 7% in HCC, HCA and HCM respectively. Statistically significant differences were found between AgNORs counts in carcinomas and adenomas (p<0.01), HCCs and HCMs (p<0.005) and betwveen NORDS in all groups (p<0.001).     

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).     

Manual versus image analysis estimation of PCNA in breast carcinoma

OBJECTIVE: To compare manual to image analysis estimation of proliferating cell nuclear antigen (PCNA) expression in paraffin sections of breast carcinomas. STUDY DESIGN: Paraffin sections of 51 breast carcinomas were stained with primary antibody to PCNA. Nuclear PCNA expression in 100 randomly selected tumor cells from marked areas was manually graded from 0 to 3. Antigen expression was also calculated by a cell analysis system (CAS-200, Becton Dickinson, Elmhurst, Illinois, U.S.A.) from marked and random microscopic fields. Obtained proliferative index (PI) from both methods was compared. RESULTS: Manually calculated PI correlated strongly with the CAS-200-calculated PI (P < .01). The highest correlation was seen between the CAS-200 PI value and manually calculated PI value using grade 2 and 3 nuclei. A particularly high correlation was noted between the number of positive nuclei and antigen staining area (P < .01) as estimated by the CAS-200. CONCLUSION: Nuclear expression of PCNA and other nuclear antigens can be accurately evaluated by an image analysis system. The speed and objectivity of such machines allow the evaluation of larger parts of tissues and provide more-representative antigen expression profiles.     

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.     

Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA.

Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase-delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies. By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti-PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme-linked immunosorbent assay (ELISA). The binding of anti-PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells. The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml-1 (protein concentration, 6.8 mg ml-1), but that of the root tip stem including the growing point increased to 887 ng ml-1 (protein concentration 3.8 mg ml-1) in the second day after germination.     

乳腺癌AgNOR计数与癌胚抗原的免疫组化研究

应用核仁组成区嗜银蛋白（ＡｇＮＯＲ）技术及癌胚抗原（ＣＥＡ）免疫组化染色对９８例乳腺良、恶性病变进行对比研究。结果表明：ＡｇＮＯＲ计数与肿瘤增殖活跃程度及生物学行为是一致的。乳腺癌中ＡｇＮＯＲ计数显著高于良性病变（Ｐ＜０．０１）。浸润癌ＡｇＮＯＲ计数比其他类型乳腺癌高。ＣＥＡ染色在乳腺良性病变中基本阴性,恶性病变中阳性率８０．８％。乳腺癌中ＡｇＮＯＲ计数与ＣＥＡ分布之间呈线性相关（ｒ＝０．８２,Ｐ＜０．０５）。ＣＥＡ阳性乳腺癌组与ＣＥＡ阴性组ＡｇＮＯＲ计数差异显著（Ｐ＜０．０５）。提示：ＡｇＮＯＲ定量研究和ＣＥＡ分布在乳腺良、恶性病变的鉴别及肿瘤恶性程度的研究中具有相似的参考价值。