Silver-stained Nucleolar Organizer Regions Research Articles

Morphologic typing of papillary renal cell carcinoma: Comparison of growth kinetics and patient survival in 66 cases

Whereas papillary renal cell carcinoma is now established as a subtype of renal cell neoplasia, division of these tumors into 2 distinctive morphotypes has been proposed. Type 1 tumors have cells with scanty pale cytoplasm arranged in a single layer on the basement membrane of papillary cores. In these tumors, psammoma bodies and foamy macrophages are frequently seen, and the tumors frequently express cytokeratin 7. Type 2 tumor cells have pseudostratified nuclei and usually have voluminous eosinophilic cytoplasm. Recent studies have supported this subclassification of papillary renal cell carcinoma by demonstrating differing genotypes for type 1 and 2 tumors. To further study the subclassification of papillary renal carcinoma, we compared clinical features, nuclear grade, stage, tumor growth kinetics, and survival in a series of 50 type 1 and 16 type 2 papillary renal cell carcinomas. Comparison of patient age at presentation, sex, and primary tumor size shows no significant difference between the 2 tumor types. Type 1 tumors were of significantly lower Fuhrman grade (P =.0001) and higher Robson stage (P =.009) than type 2 tumors. There was no significant difference when tumors were staged according to the TNM classification. Assessment of tumor growth kinetics showed significantly different mean silver-staining nucleolar organizer region (AgNOR) scores and Ki-67 indices (AgNOR type 1, 3.83, type 2, 7.24, P =.0001; Ki-67 type 1, 3.17%, type 2, 6.01%, P =.0002). Multivariate analysis showed tumor type (P =.03), presence of metastases (P =.04), AgNOR score (P =.001), and Ki-67 index (P =.03) to be independently associated with survival. These results provide evidence of the clinical utility of dividing papillary renal cell carcinomas into 2 types according to histologic characteristics. HUM PATHOL 32:590-595. Copyright © 2001 by W.B. Saunders Company

Expressions of proliferation markers (Ki-67, proliferating cell nuclear antigen, and silver-staining nucleolar organizer regions) and of p53 tumor protein in gestational trophoblastic disease

Objective: This study was undertaken to determine whether the expressions of 3 proliferation markers (Ki-67, proliferating cell nuclear antigen, and silver-staining nucleolar organizer regions) and of p53 tumor protein could differentiate spontaneous abortions from gestational trophoblastic diseases and also discriminate among gestational trophoblastic disease subgroups. Study Design: Twenty-two partial hydatidiform moles, 17 complete hydatidiform moles, 6 invasive hydatidiform moles, and 20 nonhydropic spontaneous abortions (control group) were evaluated by means of immunohistochemical techniques with antibodies to Ki-67, proliferating cell nuclear antigen, and p53. One-step silver staining was used to detect silver-staining nucleolar organizer regions. Results: The expressions of Ki-67, proliferating cell nuclear antigen, silver-staining nucleolar organizer regions, and p53 were significantly higher in the gestational trophoblastic disease group than in the control group. The results of linear discriminant analysis showed that silver-staining nucleolar organizer region count had the highest sensitivity and specificity (93.3% and 100%, respectively) for distinguishing gestational trophoblastic disease from spontaneous abortion. Sensitivity and specificity for discriminating gestational trophoblastic disease from spontaneous abortion increased to 100% when all four markers were used together. Proliferating cell nuclear antigen was found to be the best discriminating variable for differentiating among gestational trophoblastic disease subgroups. Conclusion: Our findings suggest that expressions of Ki-67, proliferating cell nuclear antigen, silver-staining nucleolar organizer regions, and p53 may aid in the diagnosis of gestational trophoblastic diseases. These fairly rapid, simple, and economic techniques could serve as a useful adjunct to conventional methods in the diagnosis of gestational trophoblastic diseases. (Am J Obstet Gynecol 2001;184:567-74.)

Effects of Green Tea on Colonic Aberrant Crypt Foci and Proliferative Indexes in Rats

The present study was designed to investigate the effect of green tea on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) formation in Wistar rats. Forty-five male weanling Wistar rats were randomly divided into three groups. Rats in Group 1 were injected with DMH (20 mg/kg sc) once a week for 10 weeks. Animals in Group 2 received 2% green tea water extract as the sole source of drinking fluid in addition to the same treatment used for Group 1. Group 3 was the negative control group. Animals were killed at the end of Week 16 after the first DMH treatment. ACF were formed in animals in their DMH-treated groups at the end of Week 16. Group 2 had fewer ACF than Group 1. Compared with the positive control group, proliferating cell nuclear antigen labeling index, silver-stained nucleolar organizer regions, and ras-p21 expression were significantly reduced in Group 2. It was concluded that green tea drinking inhibited ACF formation in rats, and such effects may be related to the suppression of cell proliferation in the intestinal crypts.

Satellite Nucleoli in the Megakaryocytic Lineage of Rats

The present study was undertaken to provide more information on the incidence of satellite nucleoli in developmental stages of the megakaryocytic lineage. Satellite nucleoli representing solitary silver stained nucleolus organizer regions (AgNORs) present in nuclei in addition to other nucleolar types were observed in all stages of megakaryocytic development. However, the incidence of satellite nucleoli was more frequent in mature megakaryocytes than in less differentiated immature megakaryoblasts and naked megakaryocytic nuclei representing the terminal stages of megakaryocytic development after loss of the cytoplasm transformed to thrombocytes. There is a possibility that the increased incidence of satellite nucleoli in mature megakaryocytes might be due to the loss of AgNORs from active nucleoli characteristic for immature cells. The decreased incidence of satellite nucleoli in naked megakaryocytic nuclei might reflect their disintegration in the terminal stages of the megakaryocytic development.

The effect of desalivation on 4-nitroquinoline-1-oxide-induced tongue carcinogenesis: a morphometric study of nucleolar organizer regions.

Saliva is an important factor in the oral cavity and could be significant in protecting against carcinogens. In experimental models of carcinogenesis, saliva was shown to have a temporary protective effect against the carcinogens DMBA and 4-nitroquinoline-oxide (4NQO). Silver-stained nucleolar organizer regions (AgNORs) are considered markers for both proliferative capacity and prognosis. The purpose of the present study was to investigate the effect of desalivation on AgNOR parameters in lesions induced by the carcinogen 4NQO in a rat model, in order to trace early nuclear changes. The study group consisted of 120 male Wistar-derived rats. The experimental group (n=56) underwent surgical desalivation; the control group (n=56) underwent a sham operation, and both groups were administered a solution of 0.001% 4NQO in the drinking water. A normal group (n=8) did not receive surgery and drank tap water. Rats were sacrificed at 7, 14, 22, and 28 weeks. Formalin fixed, paraffin embedded sections of the tongue were silver-stained and AgNOR parameters were analyzed using computerized image analysis. In both desalivated and control groups, the nuclear area was significantly higher than the normal. This difference was already evident at 7 weeks. The mean AgNOR area was significantly higher in the desalivated group at week 7 and continued to increase over time. The mean AgNOR number was also significantly higher in the desalivated group at week 7. Differences between the desalivated and control groups diminished with time. These changes in proliferative activity, as expressed by AgNOR parameters, presented earlier changes in comparison to those observed in microscopic examination of the same slides. Results suggest that saliva in the oral cavity can delay malignanttransformation, but continued exposure to the carcinogen overrides this effect. AgNOR stain seems to be sensitive and allows for early identification of intranuclear changes.

Correlation between silver-stained nucleolar organizer region area and cell cycle time.

The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units.

Prognostic Importance of the Nucleolar Organizer Region Score in Ovarian Epithelial Tumors

Objective: The score of the silver-stained nucleolar organizer region (AgNOR) is frequently found to be high in malignant tumors. We studied AgNOR in ovarian epithelial tumors diagnosed in our hospital between 1993 and 1998. Materials and Method: In our study 67 ovarian epithelial tumors (18 cystadenoma, 16 borderline type, 34 cystadenocarcinoma) were stained using the method previously described by Crocker. One-way ANOVA and logistic regression tests were used to find the statistical relationship between grade, recurrence, mortality rates and AgNOR scores in tumors (p values of <0.05 were considered statistically significant). Results: The mean AgNOR scores of 28 mucinous and 39 serous ovary tumors were calculated. The lowest AgNOR score of 1.33 was found in cystadenomas and the highest AgNOR score of 4.92 was found in serous and mucinous cystadenocarcinomas. In addition the relationship between mortality rate, recurrence and AgNOR score in carcinomas were studied. Conclusion: AgNOR scores in carcinomas were found to be higher than adenomas, and the highest AgNOR score was found in grade-III carcinomas. This shows that the AgNOR score can be used as a prognostic index in malignancies.

The 5-aminolaevulinic acid-based photodynamic effects on nuclei and nucleoli of HL-60 leukemic granulocytic precursors

To provide more information on the 5-aminolaevulinic acid (ALA)-based photodynamic effect (PDE) on nuclei and nucleoli of individual leukemic cells, these structures were studied in cultured HL-60 cells which originated from leukemic highly immature and less differentiated precursors of granulocytes. The nuclear morphology was visualized by panoptic May-Grünwald/Giemsa staining and cytochemical method for DNA, nucleoli were visualized by cytochemical methods for the demonstration of RNA and silver stainable proteins including those of interphase silver stained nucleolus organizer regions (AgNORs). In most cells ALA-based photodynamic treatment (PDT) produced marked alterations such as formation of apoptotic bodies, and large condensation of nuclear chromatin structure but without nuclear segmentation. Such changes are in harmony with the apoptotic process induced in these cells but without previous terminal differentiation. In nucleoli ALA-based PDT produced the reduction and disappearance of nucleolar silver stainable particles (SSPs) representing AgNORs which apparently reflected the alteration of the nucleolar biosynthetic activity and cell proliferation. The latter is also reflected by the disappearance of mitotic divisions. On the other hand, a small subpopulation of cells was less sensitive or resistant to the ALA-based PDE since they did not show mentioned nuclear and nucleolar alterations.

Relationship between FIGO grade and AgNOR, S100-positive langerhans cells in endometrial adenocarcinoma.

Cell kinetic information is an important adjuvant to histological grading in some malignant tumors. In the current study, 20 endometrial adenocarcinomas were graded according to the 1988 International Federation of Gynecology and Obstetrics (FIGO) and nuclear grading system and the expression of silver-stained nucleolar organizer regions (AgNOR) and S100-positive Langerhans cells were determined. According to the FIGO grade, eight patients were in grade 1, seven were in grade 2 and five were in grade 3. Nuclear grade 1, 2 and 3 was determined in six, eight and six cases, respectively. Although an overlap was seen in some grade 2 and 3 cases, AgNOR counts were the highest in grade 3. S100-positive Langerhans cells were depleted when the grades elevated. It is concluded that AgNOR and Langerhans cells can be correlated with grades. Further studies, including survival data of the patients, could be performed and AgNOR and Langerhans cells can be correlated with prognosis.

Low-grade intraosseous osteosarcoma in Northern Japan: Advantage of AgNOR and MIB-1 staining in differential diagnosis

Low-grade intraosseous osteosarcoma is an uncommon form ofbone cancer. It is occasionally difficult to recognize as a malignant tumor and is commonly misdiagnosed as a benign fibrous lesion. We retrospectively studied the records of 8 patients with low-grade intraosseous osteosarcoma in the files of the Tohoku Musculoskeletal Tumor Society in Japan. All tumors arose in the lower limb. The most common symptom was pain, with a duration exceeding 2 years in 4 patients. Radiologic findings, including those at magnetic resonance imaging (AM), suggested malignancy in 5 lesions, whereas 3 were diagnosed as benign. Two patients initially presented with pathological fracture. The initial pathological diagnosis was malignant in 5 patients and benign in 3. All eight tumors were grade 1 in Broders' classification. The tumor showed a permeative pattern in all eight cases, but this pattern could not be confirmed in the multiple tiny fragments obtained as biopsy specimens in 3 cases. The number of silver-staining nucleolar organizer regions (AgNOR) per nucleus and MIB-1-positive rate were significantly higher in low-grade intraosseou osteosarcoma than in fibrous dysplasia, offering an advantage in differential diagnosis. Three patients (38%) developed high-grade sarcoma at the site of local recurrence after multiple intralesional excisions, and one of them died of the disease. The other 5 patients had a good clinical course after surgery with a wide margin. These findings indicate that preoperative diagnosis with radiologic investigation, including magnetic resonance (MR) imaging and histologic examination of biopsy specimens is essential in preparation for surgery with a wide margin, assuring a good clinical course, and the results of AgNOR and immunohistochemical MIB-1 staining might be helpful in differentiating low-grade intraosseous osteosarcoma from fibrous dysplasia.

AgNOR are sensitive markers of radiation lesions in squamous epithelia.

The possibility of detection of incipient cellular alterations is central to early diagnosis and to clinician's capacity to discriminate between samples that appear similar on routine preparations. We examined the value of silver-stained nucleolar organizer regions (AgNOR) in detecting radio-induced alterations in a model of squamous epithelium biologically similar to oral mucosa. Morphometry of AgNOR has been proven to be of value in the detection of incipient cellular alterations. This method allows for the quantitative evaluation of lesions induced by high doses of radiation long before they become apparent in routine preparations. We herein examine the capacity of AgNOR to reveal the response to low doses of radiation, closer to the therapeutic or accidental dose to which the epithelium of oral mucosa may be exposed.

Uterine smooth muscle tumors: utility of classification by proliferation, ploidy, and prognostic markers versus traditional histopathology.

Accurate categorization of uterine smooth muscle neoplasms by light microscopic examination is difficult. Multiple classification schemes have been proposed based on mitotic rate, nuclear atypia, and the presence or absence of necrosis. None of these classification systems has been entirely successful. Multiple ancillary techniques have been tested for their ability to predict behavior of uterine smooth muscle tumors. We assayed 45 smooth muscle neoplasms for a variety of proliferation markers, oncogene protein products, and DNA ploidy level to determine if these markers supplied prognostically useful information over and above that obtained by routine light microscopic assessment. Forty-five uterine smooth muscle neoplasms were assessed for DNA ploidy; silver-staining nucleolar organizer regions (AgNORs); percent nuclear proliferating cell nuclear antigen (PCNA); expression of p53, Her-2/neu, and MDM-2 protein; mitotic rate; and nuclear grade. These markers were correlated with histologic diagnosis and the occurrence of a clinically adverse event (death, metastasis, or recurrence). Diagnostic category (P <.001), nuclear grade (P <.002), mitotic activity (P <.001), mean AgNORs (P <.001), percent nuclear PCNA (P =.02), and expression of p53 (P =.02) all correlated with clinical outcome. No statistically significant correlation between clinical outcome and the categories MDM-2 expression, Her-2/neu expression, or DNA ploidy was seen. Nuclear grade, p53 expression, mitotic rate, AgNORs, and percent nuclear PCNA correlated with diagnosis. Diagnostic category, mitotic rate, AgNOR counts, PCNA, and p53 expression dichotomized uterine smooth muscle neoplasms into prognostically favorable and unfavorable groups. Although highly significant, the category AgNORs was no more successful than mitotic rate in dividing uterine smooth muscle neoplasms into prognostically favorable and unfavorable groups. Expression of p53 and percent nuclear PCNA dichotomized uterine smooth muscle neoplasms into prognostic groups, but neither technique reached the level of significance achieved by mitotic rate. Our data indicate that mitotic rate and the classification system of Kempson and Bari are at least as effective as the tested markers in separating uterine smooth muscle neoplasms into prognostic categories.

Mutations of p53 tumor suppressor gene, apoptosis, and proliferation in intrahepatic cholangiocellular carcinoma of the liver.

This study was performed to examine the correlation between mutations of the p53 tumor suppressor gene, the occurrence of apoptosis, and proliferation in cholangiocellular carcinoma of the liver. The results obtained were compared with pathohistological stage (according to UICC) and grade and with disease related survival rate. In 41 curatively (R0-) resected intrahepatic cholangiocellular carcinomas, the status of the p53 gene was determined by direct sequencing of exons 4-9 and immunohistochemically. Apoptosis was assessed using the in situ end labeling (ISEL) technique in combination with morphological criteria. Proliferation was analyzed by immunohistochemistry of MIB-1 (Ki-67), Proliferating cell nuclear antigen (PCNA), and silver-stained nucleolar organizer regions (AgNOR). The results obtained were compared with pathohistological stage (according to UICC), grade, several other histopathological factors, and survival rate. Mutations of p53 were detected in 15/41 carcinomas examined (37%). The most common change was a G-->C and C-->T transition, changing the hot spot amino acid determined by exons 4-8. Of these 15 tumors, 14 were also p53-positive by immunohistochemistry. In each carcinoma examined, we could demonstrate MIB-1, PCNA, and AgNOR dots and also apoptotic cells in variable proportions. The proliferation markers showed a significant correlation among themselves. In univariate survival analysis, the extent of the primary tumor, lymph node status, grade, and p53 were significant factors influencing patient survival. Performing multivariate Cox regression survival analysis, however, only the extent of primary tumor and lymph node status had an independent prognostic impact. Apoptosis was not related to patient prognosis or to other parameters examined. In conclusion, these results indicated that p53 could serve as an additional prognostic parameter that could provide auxiliary information for patient outcome. However, tumor stage and lymph node involvement were the strongest prognostic factors. We failed to establish apoptosis or other pathological parameters as factors predicting the prognosis of patients with cholangiocellular carcinoma.

Silver-Staining Nucleolar Organizer Region Quantification in Pituitary Adenomas.

Nucleolar organizer regions (NORs) are segments of DNA, encoding for ribosomal RNA. They are associated with argyrophilic proteins and, thus, they can be localized through silver staining. A correlation has been shown between the number, the size, or the intranuclear localization of AgNORs, and the proliferative activity of cells. The aim of this study was to examine numerous features of AgNORs in pituitary adenomas and to relate them to immunohistochemical typing of tumor. Histologic slides from 32 pituitary tumors and one normal pituitary were silver-stained and analyzed with a computerized system for microscopic image analysis, supported by an AgNORmeter95 program. All the tumors were also immunocytochemically characterized. We have found that gonadotropinomas, when compared with pleurihormonal adenomas, revealed a lower proportion of nuclei with a single AgNOR and a higher percentage of marginal dots. Recurrent adenomas, when compared with primary adenomas, showed a higher proportion of nuclei with three AgNOR dots, a larger total area of dots in the nuclei, and a higher standard deviation of the AgNOR dot area in the nucleus. Adenomas immunopositive for prolactin, when compared with immunonegative ones, showed a larger mean area of the AgNOR dot, a larger area of the biggest dot in the nucleus, and a higher proportion of nuclei within a single dot. These results suggest that the estimated parameters of AgNOR dots differ according to tumor aggressiveness and to the hormone immunopositivity of pituitary adenomas.

Suppression of azoxymethane-induced colonic aberrant crypt foci by a nitric oxide synthase inhibitor

Nitric oxide synthase (NOS), an important bioregulator of a variety of biological processes, is overexpressed in colonic tumors of humans and rodents. In this study, effects of l-N G-nitroarginine methyl ester (L-NAME), a NOS inhibitor, on development of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in F344 male rats were investigated. Six-week-old male F344 rats were fed diets containing 0 or 100 ppm L-NAME, and given s.c. injections of AOM at 15 mg/kg body wt. once a week for 2 weeks. At 17 weeks of age, all animals were sacrificed and their colons were evaluated for numbers of ACF. Feeding of 100 ppm L-NAME inhibited the development of ACF in different sizes by 24–39%, those containing four or more crypts being most markedly affected. Assessment of silver-stained nucleolar organizer regions protein (AgNORs)/nucleus further revealed a 44% reduction by administration of L-NAME. These results suggest that the NOS inhibitor, L-NAME, may be an effective chemopreventive agent against colon carcinogenesis due to depression of cell proliferation.

Prognostic relevance of AgNORs in tumor pathology

The importance of the analysis of the silver-stained nucleolar organizer regions (AgNORs) for prognostic purposes in tumor pathology has been reviewed. Current available data from the literature demonstrate that the evaluation of the quantity of interphase AgNORs is an independent prognostic factor in several types of human tumors. Results of our investigations indicate that AgNORs are the most powerful variable predicting survival in patients with pharyngeal carcinoma, multiple myeloma, male breast and prostate carcinoma. The combination of AgNOR counts and histologic pattern allows the stratification of patients with multiple myeloma, pharyngeal and prostate carcinoma into low- and high-risk groups, which could benefit from different therapy. Moreover, AgNOR analysis predicts response to treatment in adult patients with acute myelogenous leukemia, and appears as an independent prognostic factor in a prospective study on renal cell carcinoma. Therefore, AgNOR analysis is a really important prognostic factor for several human neoplasias. The experimental and theoretical justifications for AgNORs as a prognostic factor are also reviewed, in particular the strict correlation between AgNOR quantity and tumor cell doubling time. Lastly, the lack of prognostic significance of AgNOR analysis in some circumstances is critically discussed.

Tea Preparations Protect Against DMBA-Induced Oral Carcinogenesis in Hamsters

The preventive effects of green tea, tea pigments, and mixed tea (a composite of whole water extract of green tea, tea polyphenols, and tea pigments) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in golden Syrian hamsters were studied. The right buccal pouches of animals were topically treated with 0.5% DMBA three times per week for 15 weeks. Animals were killed separately after 6, 10, and 15 weeks of DMBA treatment. Oral tumors were counted and measured, and mean tumor burden was calculated. The incidence of preneoplastic lesions and micronucleated cells, the number and volume of silver-stained nucleolar organizer regions (AgNOR), the labeling index of proliferating cell nuclear antigen, and the level of epidermal growth factor receptor expression were studied. The results showed that oral administration of 1.5% green tea, 0.1% tea pigments, and 0.5% mixed tea as the sole source of drinking water for two weeks before initiation of the DMBA treatment and until the end of the experiment significantly reduced the mean tumor burden and the incidence of dysplasia and oral carcinoma (p < 0.01). The frequency of micronucleated cells, the number of AgNOR, the total volume of AgNORs, the labeling index of proliferating cell nuclear antigen, and the level of epidermal growth factor receptor expression in the oral mucosal cells were also significantly reduced (p < 0.01). These results clearly indicated that tea preparations could effectively inhibit DMBA-induced oral carcinogenesis in hamsters. Protection from DNA damage and suppression of cell proliferation could be important mechanisms of the anticarcinogenic effects of the tea preparations.

Absence of tolerance to cataleptic activity of gamma-hydroxybutyric acid after subchronic administration to male mice

The number of silver-stained nucleolar organizer regions (Ag-NORs) was counted in stromal myofibroblasts in 125 cases of breast cancer. The mean (S.E.) of Ag-NORs in stromal myofibroblasts was 4.4 (0.1). The number of Ag-NORs in myofibroblasts was related to histological grade (p = 0.028), histological type (p = 0.013), tumour necrosis (p = 0.026), mitotic frequency (p = 0.091) and S-phase fraction (p = 0.032). The number of Ag-NORs in stromal myofibroblasts predicted survival with a borderline significance (x2 = 3.4, p = 0.052) and also in axillary lymph node negative tumours (X2 = 2.8, p = 0.095). The recurrence-free survival in the entire cohort (X2 = 7.1, p = 0.0075) and in axillary lymph node negative tumours (X2 = 7.1, p = 0.0078) could be predicted on the basis of the number of Ag-NORs in stromal myofibroblasts. In axillary lymph node positive tumours the number of Ag-NORs in stromal myofibroblasts had no prognostic value. In multivariate analysis the number of Ag-NORs in stromal myofibroblasts had no independent prognostic value.The results suggest a close interaction between stromal myofibroblasts and epithelial cancer cells in breast cancer, and stromal changes are clearly a subject for further analyses.

Nimodipine attenuates psychotigenic effects of ketamine in humans

The number of silver-stained nucleolar organizer regions (Ag-NORs) was counted in stromal myofibroblasts in 125 cases of breast cancer. The mean (S.E.) of Ag-NORs in stromal myofibroblasts was 4.4 (0.1). The number of Ag-NORs in myofibroblasts was related to histological grade (p = 0.028), histological type (p = 0.013), tumour necrosis (p = 0.026), mitotic frequency (p = 0.091) and S-phase fraction (p = 0.032). The number of Ag-NORs in stromal myofibroblasts predicted survival with a borderline significance (x2 = 3.4, p = 0.052) and also in axillary lymph node negative tumours (X2 = 2.8, p = 0.095). The recurrence-free survival in the entire cohort (X2 = 7.1, p = 0.0075) and in axillary lymph node negative tumours (X2 = 7.1, p = 0.0078) could be predicted on the basis of the number of Ag-NORs in stromal myofibroblasts. In axillary lymph node positive tumours the number of Ag-NORs in stromal myofibroblasts had no prognostic value. In multivariate analysis the number of Ag-NORs in stromal myofibroblasts had no independent prognostic value.The results suggest a close interaction between stromal myofibroblasts and epithelial cancer cells in breast cancer, and stromal changes are clearly a subject for further analyses.

Potentially malignant and malignant lesions of the lip. Role of silver staining nucleolar organizer regions, proliferating cell nuclear antigen, p53, and c-myc in differentiation and prognosis.

The cellular changes leading to carcinoma of the lip are still not completely understood. This study was carried out on 44 malignant and potentially malignant lesions of the lower lip [30 squamous cell carcinomas (SCC), 7 actinic cheilitis, 3 leukoplakias, and 4 nodal metastases from lower lip SCC]. Silver-stained nucleolar organizer regions (AgNORs) and the immunohistochemical expression of proliferating cell nuclear antigen (PCNA), p53, and c-myc were evaluated on formalin-fixed, paraffin-embedded sections. The results indicate that the size and numbers of AgNORs and the percentage of PCNA-positive cells are sensitive parameters for discriminating between potentially malignant lesions and SCC, and for the prognostic sub-typing of lower lip SCC. Furthermore, while p53 positivity was found more frequently in high-grade carcinomas, p53-positive cellular clones were also found in some potentially malignant lesions, a finding probably related to ultraviolet-related cellular damage. These p53-positive lesions could be considered at higher risk of progression to malignancy than the p53-negative ones, although there is no evidence for this as yet. c-myc positivity was found only in some high-grade carcinomas and metastases, and appeared correlated with the later phases of lip carcinogenesis. The combined evaluation of the proliferation status, together with the changes in p53 and c-myc oncoproteins, might constitute useful markers for the prognostic evaluation of potentially malignant, as well as malignant, lesions of the lip.