Nucleolar organizer regions (NORs) in simple and proliferating trichilemmal cysts (pilar cysts and pilar tumors)

Simple trichilemmal cysts or pilar cysts and pilar tumors are relatively rare entities often under-reported by pathologists. The pilar cysts are thought to proliferate and progress to pilar tumors. These pilar tumors can further undergo malignant change. We analyzed 25 pilar cysts and eight pilar tumors, including three with atypia and one malignant pilar tumor, with a view to study the above progression and assess the degree of cell proliferation using the Nucleolar Organizer Regions (AgNORs). There was a progressive increase in the AgNOR count from one dot per nucleus in pilar cysts to 1.5-2 in benign pilar tumors. AgNORs in pilar tumors with atypia (2.8) was more than the benign pilar tumors but were definitely less than the malignant pilar tumors (3.5). The malignant pilar tumor showed bizarre AgNORs and cells with as many as eight to nine AgNORs. Thus AgNOR counts suggested that there is a progressive increase in the degree of cell proliferation and thereby the AgNOR staining from pilar cysts to pilar tumors. This AgNOR staining could also be used to assess the cell proliferation in case of pilar tumors with atypia where it is difficult to rule out malignancy.     

Early stages of multistep hepatocarcinogenesis: adenomatous hyperplasia and early hepatocellular carcinoma.

From a series of 320 heptocellular carcinoma (HCC) cases treated surgically, we selected small nodular lesions that had not destroyed the preexisting liver structure grossly. After excluding metastases and large regenerative nodules, 58 lesions from 41 cases were chosen. All the lesions were hypercellular. Among them, 33 lesions showing histologic features of very well-differentiated HCC (Edmondson grade I), that is, small hepatocytes with little cellular atypia but with structural atypia, such as a thin trabecular structure of acinar formation in some areas, were classified as early HCC (eHCC). In seven eHCCs, areas of overt carcinoma, classified as Edmondson grade II, were found in the background of Edmondson grade I carcinoma. The remaining 25 lesions lacked structural atypia and were classified as adenomatous hyperplasia (AH). Among the AHs, 10 nodules with a very focal abnormal structure were subclassified as atypical adenomatous hyperplasia (AAH). There was a tendency for the size and cellularity of the atypical lesions to increase in order from AH to AAH to eHCC. All nodules larger than 1.5 cm were eHCC. A degree of cellularity more than twice that of a regenerative nodular was suggested to be an indicator of HCC. All small nodular lesions were associated with chronic liver disease. These histologic observations appear to explain the stepwise development of overt HCC from very well-differentiated eHCC, and of eHCC from AH probably through AAH, at least in cases of HCC associated with chronic liver disease.     

Histopathological and morphometric analysis of atypical adenomatous hyperplasia of human cirrhotic livers

Atypical adenomatous hyperplasia (AAH) is a hyperplastic parenchymal nodular change in the cirrhotic liver, in which overt hepatocellular carcinoma (HCC) occasionally arises. AAH is defined as a sizable hepatocellular nodule with a variable degree of hepatocellular atypia not regarded as HCC, and is different from ordinary adenomatous hyperplasia in which hepatocellular atypia is absent. In the present study, we attempted to evaluate carcinogenetic processes and to find histological variables which indicate malignant transformation in AAH, using 49 surgically resected or autopsied nodules. AAH frequently showed morphological heterogeneity. Atypical lesions within AAHs were divisible into the following three categories from overall histopathological appearances: malignant (A), equivocal (B), or non-malignant (C) lesions. Analysis of combination of these three lesions, which were frequently intermixed in a given AAH, suggested that B lesions appear subsequent to C lesions, and A lesions finally appear in AAH nodules. Among the 14 histological variables, enlargement, hyperchromasia and irregular contour of nuclei were found to correlate well with A lesions. Increased nuclear density, iron resistance, reduction of reticulin fibres, clear cell change, sinusoidal dilatation and presence of abnormal arteries were suggestive of A or B lesions. Nuclear deviation toward the sinusoids, acinar and compact arrangements, fatty change and Mallory's hyaline alone were not useful indicators of A or B lesions. These results indicate that AAH is a preneoplastic or borderline lesion in which overt HCC is likely to evolve through several steps. Although a needle liver biopsy is a useful tool for diagnosis of benign, equivocal and malignant hepatocellular nodular lesions, the needle biopsy specimen should be carefully evaluated by considering the morphological heterogeneity of the AAH and a variable combination of 14 histological variables.     

Role of nucleolar organiser regions in differentiating malignant from benign tumours of the colon.

An argyrophilic technique (AgNOR) was applied to paraffin wax sections of 12 tubular adenomas, 17 villous adenomas with moderate and severe atypia, and 21 colonic adenocarcinomas. The range of the mean number of nucleolar organiser regions (NORS) per nucleus was 1.54-3.28 (99% CI 2.29-3.04) for tubular adenomas 3.07-4.36 (2.98-4.43), and 3.60-5.02 (3.74-4.69) for villous adenomas with moderate and severe atypia, respectively, and 5.53-9.33 (6.15-8.54) for highly differentiated adenocarcinomas. The number of AgNORs permitted differentiation among the three groups. The differences observed were significant. Malignant tumour cells were characterised by a large number of AgNORs which were small in size and showed a scattered distribution. Nuclei of tubular adenoma and villous adenoma with moderate atypia had only a small number of large sized AgNORs in a clustered distribution. It is suggested that this method distinguishes malignant epithelial cells from benign cells of colon, even those with severe atypia, and that it is a useful adjunct to diagnostic histopathology.     

Breast carcinoma kinetics. Argyrophilic nucleolar organizer region counts correlate with Ki67 scores

This study assessed the value of argyrophilic nucleolar organizer region (AgNOR) staining as a potential technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Using a silver staining technique and immunohistochemistry, the authors correlated the numbers of argyrophilic nucleolar organizer regions (AgNORs) and Ki67 scores in 70 breast carcinomas and 27 benign breast lesions. Epithelial cells in fibrocystic disease and fibroadenomas contained a mean of 2.65-6.8 small uniform AgNORs per cell, whereas malignant cells contained 4.6-26.9 frequently highly irregular AgNORs. In benign tissue, Ki67 scores ranged from 0 to 4%; in malignant tumors, Ki67 scores ranged from 3.0 to 98%. The correlation between AgNOR counts and Ki67 scores was highly significant (P less than 0.001). The authors concluded that AgNOR counts performed on routine formalin-fixed paraffin sections furnish significant kinetic information. Furthermore, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic value.     

A Histopathological Analysis of Five Cases of Adenomatous Hyperplasia Containing Minute Hepatocellular Carcinoma

We present five cases of adenomatous hyperplasia (AH) containing minute hepatocellular carcinoma (HCC) in cirrhotic liver. All the patients were Japanese, four males and one female, ranging in age from 60 to 80 years. Two of the specimens were obtained at surgery and the others at autopsy. The AH specimens ranged from 2.0 to 3.0 cm in diameter, and the maximum diameter of HCC foci in the AH was 2.0 cm. Histologically, apart from the HCC foci, the AH specimens showed intrinsic atypia, suggesting preneoplastic change. These features included an increase of both cellularity and the nucleo cytoplasmic ratio, distortion of cord structure and pseudoacinar formation. Ail of the AH specimens contained typical portal triads. Details of diagnostic imaging were also obtained in four cases. The findings of the present study support the possibility that AH with intrinsic atypia is a preneoplastic lesion of HCC. The sequence of "adenomatous hyperplasia with intrinsic atypia HCC foci" would thus represent part of the early phase of hepatocarcinogenesis in humans.     

Nucleolar organiser regions in hyperplastic and neoplastic prostatic tissue

Summary Silver-stained nucleolar organiser regions (AgNORs) were studied in 10 hyperplastic, 5 intra-epithelial neoplastic and 30 malignant prostatic lesions. Total AgNOR counts and types were compared with histological features. The total AgNOR count per nucleus was significantly higher (p<0.05) in prostatic intra-epithelial neoplasia (PIN) and adenocarcinoma compared with prostatic hyperplasia. In addition, satellite Ag-NORs predominated in hyperplasia, while medium-sized and large nucleoli with granular AgNORs were only observed in PIN and adenocarcinoma. The results indicate that, despite statistically significant differences, Ag-NOR counts are of no use for diagnosis of any single case in the groups studied, because of considerable overlap. AgNOR typing, however, may contribute to the differential diagnosis between benign and malignant lesions. We propose a new AgNOR typing system.     

AgNOR counts are not altered in alcoholic cirrhosis

One hundred and two liver biopsy specimens were stained for Argyrophilic nucleolar organizer regions and associated proteins to assess its utility in differentiating normal, cirrhotic and neoplastic liver tissue. A statistically significant (p < 0.001) difference was observed between mean AgNOR counts of normal (1.53 +/- 0.4), post-hepatitic cirrhosis (3.65 +/- 0.53) and hepatocellular carcinoma (7.94 +/- 1.18). In contrast the mean AgNOR count of biopsies with alcoholic cirrhosis (1.57 +/- 0.06) was significantly less (p < 0.001) than post-hepatitic cirrhosis and was similar to that of normal liver tissue. It is concluded that AgNORs can act as a good adjuvant to histology in diagnosing liver diseases especially in differentiating post-hepatitic and alcoholic cirrhosis.     

STUDIES ON THE PROLIFERATIVE ACTIVITY OF DIETHYLNITROSAMINE-INDUCED HEPATOCELLULAR CARCINOMAS IN MONKEYS

In this study, synthetic phase fractions (SPFs) determined by flow cytometry and AgNOR counts were analysed in benign liver lesions (regenerative nodules and adenomas), hepatocellular carcinomas (HCCs), and lung metastases of a monkey hepatocarcinogenesis model to find out if AgNOR counts and SPFs can discriminate between malignant and non-malignant liver lesions. The average per cent SPF values and the AgNOR counts were significantly (P=0·001) increased in regenerative liver nodules (5·30 per cent; 4·96), adenomas (5·34 per cent; 3·46) and well-differentiated HCCs (6·75 per cent; 4·47), compared with the untreated control livers (3·18 per cent; 0·98), but the differences between these three groups were not significant. In the poorly differentiated HCC group, however, the average SPF value (9·60 per cent) and AgNOR count (7·14) were significantly higher than in any of the other liver lesions examined. A significant correlation was found between the SPF values and AgNOR counts on the one hand, and differentiation and cytological grade of the HCC samples on the other. The results of this study show that the SPF values and AgNOR counts are not reliable in differentiating between regenerating liver nodules, adenomas, and experimental well-differentiated HCCs. The SPF value, however, may serve as a prognostic factor in HCC, since it was found to be significantly higher in HCCs with lung metastasis than in those without. © 1997 by John Wiley & Sons, Ltd.     

A histopathological analysis of five cases of adenomatous hyperplasia containing minute hepatocellular carcinoma.

We present five cases of adenomatous hyperplasia (AH) containing minute hepatocellular carcinoma (HCC) in cirrhotic liver. All the patients were Japanese, four males and one female, ranging in age from 60 to 80 years. Two of the specimens were obtained at surgery and the others at autopsy. The AH specimens ranged from 2.0 to 3.0 cm in diameter, and the maximum diameter of HCC foci in the AH was 2.0 cm. Histologically, apart from the HCC foci, the AH specimens showed intrinsic atypia, suggesting preneoplastic change. These features included an increase of both cellularity and the nucleo-cytoplasmic ratio, distortion of cord structure and pseudoacinar formation. All of the AH specimens contained typical portal triads. Details of diagnostic imaging were also obtained in four cases. The findings of the present study support the possibility that AH with intrinsic atypia is a preneoplastic lesion of HCC. The sequence of "adenomatous hyperplasia with intrinsic atypia-HCC foci" would thus represent part of the early phase of hepatocarcinogenesis in humans.     

Morphometric study of adenocarcinomas and hyperplastic epithelial lesions in the peripheral lung

Mean nuclear areas (MNA) were calculated morphometrically in ten cases of well-differentiated adenocarcinoma, ten cases of atypical alveolar cuboidal cell hyperplasia (AAH), and five cases of alveolar cuboidal cell hyperplasia (AH) and were compared with each other. In cases of adenocarcinoma, the MNA were significantly larger than those of AAH and AH, and the standard deviation (SD) of nuclear area was greater in adenocarcinoma than that in AAH and AH, thus implying greater "scatter" of the nuclear areas in the former than the latter two. The MNA and the SD of nuclear area in AH were smallest in these three groups. In one case each of AAH and adenocarcinoma, there were two different populations of nuclear size in the same lesions, that is, histograms showed both adenocarcinoma and AAH in the same tumor. Through the use of the morphometric method, many cases of AAH were easily distinguishable from adenocarcinoma cases, and there were two cases in which foci of adenocarcinoma and AAH coexisted.     

Argyrophilic nucleolar organizer regions in Wilms' tumors and related lesions: a histologic and chromosomal correlation.

The argyrophilic nucleolar organizer regions (AgNORs) have been extensively investigated in different neoplasms, and their increased number has been frequently linked to aggressive tumor behavior. To evaluate this association, 22 specimens from 21 patients with Wilms' tumors and related lesions were stained by the AgNOR silver-staining technique in formalin-fixed, paraffin-embedded tissue. The aim of the study was to assess the significance of AgNOR count independently or in relation to histology in predicting the behavior of these neoplasms. Eleven nonneoplastic pediatric kidneys were used as controls. The controls had a range of 1.55 to 2.26 AgNOR/nucleus with a mean +/- SD of 1.91 +/- 0.22. The tumors showed a range of 1.25 to 2.86 AgNOR/nucleus with a mean +/- SD of 2.10 +/- 0.38 with obvious overlap between tumors and controls. The two unfavorable histology Wilms' tumors and the malignant rhabdoid tumor showed no increase in the number of AgNOR (1.66, 2.04, and 2.05, respectively). We further quantitated the nucleolar organizer regions (NOR) in metaphase spreads of three of the neoplasms, compared those with the patients' peripheral blood, and showed no difference in NOR numbers except for an occasional decrease in G-group NORs in the tumor cells. Tumors that had metaphase NOR counts of 6 to 9/nucleus had a mean interphase AgNOR count of less than 2.5. These results suggest that quantitative analysis of AgNORs in Wilms' tumors and related lesions is not a reliable predictor of aggressive tumor behavior. The discrepancy between metaphase and interphase NOR counts indicates that AgNOR counts are merely the reflection of the spatial arrangement of NOR-bearing chromosomes.     

AgNOR counts and determination of malignancy in stromal tumours of the stomach and small intestine.

Twenty four primary stromal tumours of the stomach and small intestine were investigated by silver staining of interphase nucleolar organiser regions (AgNORs) in an attempt to obtain an objective criterion for prediction of malignant tumour behaviour. Malignant tumours tended to have higher AgNOR counts than their benign counterparts, but this increase was small and there was some overlap between the two groups. There was a correlation between the mean AgNOR count and the mitotic count. There was no correlation between tumour size and these two measurements. This study suggests that in these stromal tumours the AgNOR count is not a useful independent predictor of malignant behaviour.     

Modified method of AgNOR staining for tissue and interpretation in histopathology

This study was conducted in the department of Pathology King Edward Medical University, from June to December 2002 to introduce the new method of AgNOR staining and its interpretation to increase its reliability. A total of 60 brain specimens were stained with modified AgNOR technique. The diagnosis of malignancy was made on H & E staining. AgNOR counts, variation in size and dispersion of AgNOR dots in cells were graded and compared in malignant and non-malignant lesions. Modified method of AgNOR staining and interpretation was an easy, reliable and reproducible alternative to traditional AgNOR techniques for evaluating proliferation activity of cells in malignant and benign brain lesions. mAgNOR counts of different grades of astrocytoma (2.97+/-0.96, 3.97+/-0.43, 6.01+/-2.74 and 8.01+/-3.56) were significantly (P<0.01) greater when compared with counts of normal brain (0.40+/-0.01), and reactive gliosis (0.60+/-0.01). AgNOR size and dispersion were of higher grade in a significantly greater proportion of malignancy when compared with benign conditions (P<0.05). The AgNOR dots were brighter and more clear with modified staining when compared with previous studies. We conclude that modified AgNOR staining technique is simple, quick and reliable to evaluate cell proliferation by detecting AgNORs size and dispersion. In future, AgNOR size and dispersion should be considered rather than the count only. We recommend the use of morphometry for AgNOR size in future. We also recommend the use of modified AgNOR staining for obtaining sound and confidant results in routine paraffin sections.     

Two AgNOR counts in fine-needle aspirates of lymphoproliferative disorders compared with acridine orange flow cytometry.

Studies have shown that argyrophilic nucleolar organizer region-associated proteins (AgNORs) may correlate with DNA ploidy and/or proliferative activity in neoplastic and non-neoplastic conditions. However, studies have estimated only the mean AgNOR counts. Here we used two AgNOR counts, one of which may correlate with DNA ploidy and the other with proliferative activity. The mean AgNOR count (mAgNOR) was defined as the mean number of AgNORs/nucleus in 100 cells and may represent DNA or RNA index. The percentage of nuclei exhibiting 5 or more AgNORs/nucleus (pAgNOR) may reflect proliferative activity. These two AgNOR counts were correlated with results from acridine orange flow cytometry in 50 fine-needle aspirate (FNA) smears of nodal and extranodal sites, including three cases of reactive lymphadenopathy and 47 cases of non-Hodgkin's lymphoma. The mean mAgNOR count in the diploid specimens was 2.03 (+/- 0.74 SD) and 2.62 (+/- 0.73 SD) in the aneuploid tumors (P less than 0.0001). Samples with a low RNA index had mean mAgNOR of 1.80 (+/- 0.41 SD), whereas those with high RNA had a mean mAgNOR of 2.93 (+/- 0.86 SD) (P less than 0.0001). Lesions with low proliferative index, determined by flow cytometry, had a mean pAgNOR of 4%, whereas those with intermediate and high proliferative indices had a mean pAgNOR of 16% (P less than 0.0001). A similar but less significant correlation existed between RI and pAgNOR (P less than 0.005). We conclude that the two AgNOR counting methods may reliably reflect cell kinetics and distinguish ploidy from proliferative activity, making them useful adjuncts to flow cytometry in limited cytology specimens and small biopsy samples.     

Nucleolar organizer regions in benign and malignant glandular lesions of the cervix.

The argyrophil technique for nucleolar organizer regions was applied to cases of normal cervix (n = 6), microglandular hyperplasia (n = 6), adenocarcinoma in situ (n = 15), and invasive adenocarcinoma of the cervix (n = 19). A rigorous staining technique was employed which facilitated the enumeration of individual AgNORs even when they were aggregated as tight clusters within the nucleolus (AgNUs). Two methods of counting AgNORs were used: a simple enumeration of dispersed AgNORs and AgNUs, and the more time-consuming counting of all individual AgNORs, including those within AgNUs. With both techniques, there was no significant difference in counts between in situ and invasive adenocarcinoma, but cases of microglandular hyperplasia showed significantly fewer AgNORs than either of these. This suggests that AgNORs may be useful in differentiating difficult cases of microglandular hyperplasia from adenocarcinoma and that the simplified counting technique is adequate for this purpose. AgNOR counts are of no use in discriminating between invasive and in situ adenocarcinoma.     

A critical evaluation of AgNOR counting in benign naevi and malignant melanoma.

There is considerable variation in the quoted mean numbers of AgNORS per nucleus for benign melanonaevi and malignant melanomas. This is partly attributable to different approaches to AgNOR counting. This study summarizes our experience in devising an optimal technique for counting AgNORs. We show that it is essential to count intra-nucleolar AgNORs in addition to those lying outside the nucleolus to obtain clear separation of naevi from melanoma. Although this seems an onerous task, we further demonstrate that a maximum of only 30 nuclei need to be counted to obtain a mean AgNOR count per nucleus which is representative of the whole lesion. This compares with the arbitrary figure of 100 nuclei chosen by most workers. Only by optimizing and standardizing all aspects of the AgNOR technique including fixation, staining, and counting will mean AgNOR counts per nucleus become a useful quick, reproducible method which can be applied to lesions which pose diagnostic problems such as borderline melanocytic lesions.     

Silver staining of nucleolar organizer regions in prostatic lesions

Variations in the number of silver-stained nucleolar organizer region-associated proteins (AgNORs) were studied in paraffin sections of 42 benign prostatic lesions, comprising four cases of granulomatous prostatitis, five of squamous or transitional metaplasia, eight of atypical and 25 of regular hyperplasia, and 37 of prostatic adenocarcinoma, with their metastases. There was a significant difference between the mean AgNOR counts of the benign and malignant prostatic lesions (1.58 +/- 0.26 v. 4.34 +/- 1.53; P less than 0.01). The mean AgNOR counts significantly increased with increasing Gleason's grade (P less than 0.01) and clinical stage (P less than 0.05) of the tumours. AgNOR counting may contribute to the conventional diagnostic and prognostic indices of cancer of the prostate.     

Nucleolar organiser regions in normal, cirrhotic, and carcinomatous livers.

A series of 54 liver biopsy specimens was studied by means of the argyrophil (AgNOR) technique for nucleolar organiser region (NOR)-associated proteins. These included normal livers and livers affected by chronic active hepatitis, cirrhosis, hepatocellular carcinoma and adenoma. Four of the cases of cirrhosis showed liver cell dysplasia. The mean numbers of NOR sites in normal, cirrhotic, and carcinomatous livers were significantly different: adenoma had similar mean counts to those in chronic active hepatitis (CAH). There was no overlap between the ranges of NOR counts in normal, cirrhotic, and malignant liver specimens. Where cirrhosis and hepatocellular carcinoma were present in the same specimen, the AgNOR counts were higher in the carcinomatous than cirrhotic areas. To investigate the prospective value of the method a further seven biopsy specimens were studied; in these it had not been possible to decide on a diagnosis between normality and cirrhosis or cirrhosis and hepatocellular carcinoma. In all seven specimens a repeat biopsy or necropsy gave results as predicted by AgNOR staining. It is therefore proposed that quantitation of staining for NOR-associated proteins is a diagnostically useful method in liver disease.     

Morphometric analysis of nucleolar organizer regions in benign and malignant peritoneal effusions using backscattered electron microscopy.

Nucleolar organizer regions are loops of DNA associated with silver-stainable proteins (AgNORs). In general, malignant cells have more and larger AgNORs than benign cells. An inconsistent argyrophilic method and difficulties in objectively evaluating AgNORs account for some of the reluctance to utilize AgNOR staining as a diagnostic tool to differentiate benign and malignant lesions. Sections from paraffin-embedded cell blocks of 10 cases of benign and malignant peritoneal effusions were stained with a modified AgNOR method. Backscattered electron imaging in the scanning electron microscope, together with image analysis, was used to evaluate more objectively a number of AgNOR parameters and to determine which measurement was the most reliable discriminant of the two types of fluids. One hundred nuclei per case were identified and imaged. In contrast to benign nuclei, AgNORs in malignant nuclei were more numerous (P less than 0.0001) and larger (P less than 0.0001). A cut-off mean AgNOR area of 1.1 microns 2 (P less than 0.0001) correctly categorized all malignant (greater than 1.1 microns 2) and benign (less than or equal to 1.1 micron 2) cases. This system's objectivity and specificity could be used to enhance the cytological interpretation of effusions, where the separation of reactive mesothelial cells and malignant cells is extremely difficult.