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.     

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.     

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。     

Epidemiological Study of Mammary Tumors in Female Dogs Diagnosed during the Period 2002-2012: A Growing Animal Health Problem

Epidemiological studies enable us to analyze disease behavior, define risk factors and establish fundamental prognostic criteria, with the purpose of studying different types of diseases. The aim of this study was to determine the epidemiological characteristics of canine mammary tumors diagnosed during the period 2002-2012. The study was based on a retrospective study consisting of 1,917 biopsies of intact dogs that presented mammary gland lesions. Biopsies were sent to the Department of Pathology FMVZ-UNAM diagnostic service. The annual incidence of mammary tumors was 16.8%: 47.7% (benign) and 47.5% (malignant). The highest number of cases was epithelial, followed by mixed tumors. The most commonly diagnosed tumors were tubular adenoma, papillary adenoma, tubular carcinoma, papillary carcinoma, solid carcinoma, complex carcinoma and carcinosarcoma. Pure breeds accounted for 80% of submissions, and the Poodle, Cocker Spaniel and German Shepherd were consistently affected. Adult female dogs (9 to 12 years old) were most frequently involved, followed by 5- to 8-year-old females. Some association between breeds with histological types of malignant tumors was observed, but no association was found between breeds and BN. Mammary tumors in intact dogs had a high incidence. Benign and malignant tumors had similar frequencies, with an increase in malignant tumors in the past four years of the study. Epithelial tumors were more common, and the most affected were old adult females, purebreds and small-sized dogs. Mammary tumors in dogs are an important animal health problem that needs to be solved by improving veterinary oncology services in Mexico.     

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.     

Nucleolar organizer region-associated proteins in cancer cells. Quantitative investigations on gliomas, meningiomas, urinary bladder carcinomas and pleural lesions.

Using a modified silver staining technique, we investigated nucleolar organizer region-associated proteins (AgNORs) in paraffin sections of 156 neoplastic tissues and other lesions, including gliomas (n = 41), meningiomas (n = 20), urinary bladder carcinomas (n = 58), and neoplastic and reactive lesions of the mesothelium of the pleural cavity (n = 37). We found significant differences in the mean number and area of AgNORs per nucleus between nonanaplastic and anaplastic astrocytomas. In meningiomas AgNOR analysis may be useful to distinguish between mostly benign tumors (grade 1 tumors) and atypical ones. Urinary bladder carcinomas exhibited a statistically significant increase in both AgNOR number and area as the grade of malignancy increased. Diagnostically useful differences in the AgNOR configuration between inflammatory and neoplastic processes were found for mesothelial lesions. In general, a higher grade of malignancy correlated with an increase in the AgNOR number. This was accompanied by an increase in the total AgNOR area per nucleus, irrespective of whether the size of the individual AgNORs had changed.     

Melanin bleaching in argyrophilic staining of AgNORs in pigmented lesions: a morphometric evaluation

OBJECTIVE: To establish a procedure that can effectively bleach melanin from pigmented lesions without affecting quantification of argyrophilic staining of nucleolar organizer regions (AgNORs). STUDY DESIGN: Twenty banal compound nevi, five from each of nonpigmented, slightly pigmented, moderately pigmented and heavily pigmented groups, were bleached by 10% H202 for periods of 0 (nonbleached controls) and 24 hours. AgNOR size and count parameters of nevomelanocytic nuclei were measured by video image analysis. Melanin bleaching using KMnO4 was also investigated. RESULTS: In all lesions treated with 10% H202 for 24 hours, the melanin was bleached effectively, with no qualitative change in AgNOR appearance. There were no significant differences in mean AgNOR number per nucleus (AgNOR number), mean individual AgNOR size (AgNOR size) or mean percentage of AgNOR area per nucleus (% nuclear area) between nonbleached and bleached sets in both the nonpigmented and slightly pigmented groups. However, disintegration of AgNOR dots was observed in those treated with 1% KMnO4 for 5, 10 and 15 minutes. There were significant decreases in AgNOR size (P = .002) and % nuclear area (P = .003) and increase in AgNOR number (P = .05) in the slightly pigmented group evaluated when treated with 1% KMnO4 for five minutes. CONCLUSION: Melanin in pigmented lesions can be bleached effectively with an H202 procedure without significantly affecting AgNOR staining properties in contrast to bleaching with KMnO4.     

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.     

Multiparameter analysis of AgNOR in thyroid lesions: comparison with PCNA expression

The aim of the study was to examine numerous features of argyrophilic proteins related to nucleolar organizer regions (AgNORs) in thyroid tumors, relate them to PCNA expression and evaluate which of these features might be useful in the diagnosis of thyroid lesions. Paraffin sections of 100 thyroid tumors were silver-stained and divided into 9 groups: nodular goiter (NG), simple adenoma (SA), microfollicular adenoma (MFA), follicular carcinoma (FC), follicular variant of papillary carcinoma (PC-F), classical variant of papillary carcinoma (PC-C), Hürthle cell adenoma (HA), Hürthle cell carcinoma (HC), and anaplastic carcinoma (AC). The slides were analyzed with the computerized system for image analysis. A weak correlation was found between PCNA expression and AgNOR size. AC differed significantly from all other examined groups in many features of AgNOR dots. Hürthle cell neoplasms were characterized by the presence of a usually single and relatively large dot. With respect to diagnosing follicular lesions, we found that the evaluation of the total area of dots in the nucleus seemed to be the most useful for discrimination: the assumption of 4.9 micro m2, as a cut-off value, allowed a correct classification of 77% of FC cases. Computer-aided morphometric analysis of AgNORs may be useful in the diagnostics of thyroid lesions.     

膀胱肿瘤DNA含量、核形态学特点与AgNORs计数的研究

应用图像分析技术对４３例膀胱肿瘤细胞ＤＮＡ含量和核形态参数进行了定量测定,同时进行核仁组成区（ＡｇＮＯＲｓ）计数研究,结果表明：肿瘤细胞的ＤＮＡ含量及干系水平与组织学分级基本一致,ＤＮＡ含量及干系水平越高,则恶性程度越高,ＤＮＡ含量与核面积及核最短直径的变化里正相关性,与ＡｇＮＯＲｓ的计数无明显相关性。这三项检测方法对肿瘤生物学行为判断均有重要的参考价值。     

Mammary gland tumors in captive African hedgehogs

From December 1995 to July 1999, eight mammary gland tumors were diagnosed in eight adult captive female African hedgehogs (Atelerix albiventris). The tumors presented as single or multiple subcutaneous masses along the cranial or caudal abdomen that varied in size for each hedgehog. Histologically, seven of eight (88%) mammary gland tumors were malignant. Tumors were classified as solid (4 cases), tubular (2 cases), and papillary (2 cases). Seven tumors had infiltrated into the surrounding stroma and three tumors had histologic evidence of neoplastic vascular invasion. Three hedgehogs had concurrent neoplasms. These are believed to be the first reported cases of mammary gland tumors in African hedgehogs.     

Morphometric image analysis of follicular lesions of the thyroid

OBJECTIVE: To determine the role of image morphometry in distinguishing various follicular lesions of the thyroid in cytologic smears. STUDY DESIGN: Archival fine needle aspiration smears of 10 cases each of follicular hyperplasia, follicular adenoma, follicular carcinoma and follicular variant of papillary carcinoma were used for the study. All cases were histopathologically proven. At least 100 random nuclei from each case were subjected to analysis with an image cytometer. Area, convex area, length, width, perimeter, convex perimeter and roundness of nuclei were measured using a 40 x objective (1 pixel = 0.446 micron). RESULTS: ANOVA showed that all the nuclear variables studied were significantly different (P < .05) in follicular hyperplasia as compared to follicular carcinoma and papillary carcinoma. All nuclear variables except roundness were also significantly different (P < .05) between follicular hyperplasia and follicular adenoma. However, between follicular adenoma, follicular carcinoma and papillary carcinoma there was considerable overlap of nuclear morphometric parameters. CONCLUSION: Image morphometry may help to distinguish nonneoplastic follicular lesions (hyperplasia) from neoplastic lesions (adenomas and carcinomas). However, to distinguish benign from malignant follicular lesions, image morphometry might not improve the accuracy of standard cytologic examination.     

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.     

Application of morphometric methods to the cytologic study of intradermal nevi.

Twenty-one intradermal nevi were studied by morphometric methods in an attempt to morphologically characterize the two types of nevus cell--epithelioids, type A, and fusiforms, type C--and to quantify the differences between them. Morphometric parameters of the intradermal nevi were compared with similar parameters of melanocytes and melanoma cells so that the maturation rates of the nevi cells could be established and to see if the parameters might indicate the degree of malignancy. Superficial nevus cells were differentiated from deep cells by their larger size and larger nuclear area. Nuclear area appeared to have potential for differentiating benign from malignant tumors. Decrease in cellular area appeared to indicate maturation rather than atrophy. Melanoma cells were differentiated by their larger size. Cell nuclear perimeter appeared to have confirmatory value, while cell perimeter was inconclusive.     

Standardized AgNOR analysis as a prognostic parameter in endometrial carcinoma, endometrioid type

OBJECTIVE: To evaluate interobserver reproducibility of histologic grade in endometrial adenocarcinomas of endometrioid type (EC), to assess the relationships between nuclear grade and the amount of argyrophilic nucleolar organizer region (AgNOR) proteins and to determine the prognostic value of AgNOR proteins and the main clinicopathologic parameters. STUDY DESIGN: Architectural and nuclear grading were independently assessed by two pathologists in 64 formalin-fixed, paraffin-embedded surgical samples of EC obtained from an equal number of patients (age range, 38-84 years; mean, 63.5). Interobserver agreement was determined using the kappa statistic; discrepant cases were reviewed, and a consensus was reached. Standardized AgNOR analysis was performed according to the guidelines of the Committee on AgNOR Quantification, measuring the mean area of AgNORs per nucleus (NORA) by an image analysis system. RESULTS: The kappa values for interobserver agreement were substantial for architectural grading and moderate for nuclear grading. When NORA values were compared to the nuclear grade assessed by different observers, the most significant linear correlation (r = .713, P < .001) was found for the nuclear assessment obtained by consensus of the two pathologists. Moreover, statistical analysis allowed discrimination of architectural grade 1 from grade 2 and 3 EC. By the Kaplan-Meier method, the prognosis was worse for patients with higher NORA values (> 4.212 micron 2), while, by Cox multivariate analysis, AgNOR quantity emerged as an independent prognostic variable. CONCLUSION: Use of standardized AgNOR analysis may be an additional and objective tool in the assessment of histologic grade as well as a reliable method of determining prognosis in EC.     

Discrimination of benign versus malignant mixed tumors of the salivary gland using digital image analysis

Seven benign and four malignant mixed tumors of the salivary gland, biopsied using fine-needle aspiration, were analyzed using digital image analysis. Mean nuclear form factor, perimeter, and area were significantly increased in malignant cases. Better separation between diagnostic categories, however, was achieved by utilizing the coefficient of variation (CV) within a case rather than mean value. Form factor CV alone divided cases into nonoverlapping diagnostic categories. This quantitative analog of "pleomorphism" provided a useful marker for malignancy in mixed tumors.