[Significance of the AgNOR in tumor pathology]. / Significato delle AgNOR in patologia tumorale.

Analysis of silver-stained nucleolar organizer regions (AgNORs), originally regarded as a diagnostic tool, is now considered mainly as a prognostic parameter. Indeed, the expression of AgNOR proteins is associated with several biological properties of neoplastic cells: metabolic activity, DNA content, histological grade of differentiation and, especially, the rapidity of cellular proliferation. Thus, a high AgNOR quantity is a marker of aggressive tumour phenotype, and a large number of papers have shown the independent prognostic value of AgNOR analysis in several human neoplasias. Moreover, the method can be applied to small biopsies, can identify neoplastic clones with different proliferative activities and may stratify patients into different risk groups. The standardized method for AgNOR quantification offers objective and reproducible results. The evaluation of AgNOR quantity in cycling cells, either by immunohistochemistry or by a novel flow cytometry technique, may represent the future of AgNOR analysis.

Nuclear morphometry in male breast carcinoma: association with cell proliferative activity, oncogene expression, DNA content and prognosis.

To investigate the prognostic value of nuclear morphometry in male breast carcinoma (MBC), histological samples from 50 patients (mean age 62.2 years) were retrospectively analyzed by computerized nuclear morphometry. All patients received surgery; 35 had multiple combinations of adjuvant therapies. Mean follow-up was 67 months (range 1-230). In each case, 100 tumor cells were measured, and the mean nuclear area (MNA), standard deviation of the nuclear area (SDNA), mean nuclear perimeter (MNP), standard deviation of the nuclear perimeter (SDNP) and shape factor (SHF) were calculated. Morphometric features were compared with tumor histological grade, size, nodal status, DNA ploidy evaluated by flow-cytometry and cell proliferative activity assessed by the quantity of argyrophilic nucleolar organizer region-associated proteins (AgNORs), monoclonal antibody (MAb) PC10 against proliferating cell nuclear antigen and MAb MIB-1. Comparison was also made with the immunohistochemical detection of p53, bcl-2, c-erbB-2 and c-myc proteins. Significant association was found between nuclear morphometric parameters and tumor grade, DNA content and cell proliferation indices. SDNA was greater in p53-positive and bcl-2-negative cases; SDNP was greater in p53-positive cases; SHF was lower in p53- and c-myc-positive cases. Overall survival was shorter in carcinomas with high MNA, SDNA, MNP and SDNP and low SHF. In multivariate analysis, performed by testing nuclear morphometric parameters, histological grade, tumor size, nodal status and p53 immunostaining in the Cox model, p53 over-expression and histological grade retained independent prognostic significance. When p53 was excluded, only SDNP appeared as an independent prognostic variable. Our results indicate that nuclear morphometric parameters can identify an aggressive tumor phenotype and provide additional prognostic information for patients with MBC.

Recurrent pelvic pain in women.

We conducted a prospective survey from January to September 1999 on a random population from the city of Turin, Italy, to highlight pain pathologies at various ages and possible differences between sexes. By means of a questionnaire, women were asked about age, profession, marital status, menstrual pain, type of delivery, number of children, onset and presence of pain of any type. Pain-related questions included its characteristics, familial tendencies, treatment, type, duration, daily and monthly quantity of medication taken, habits, previous pathologies, or surgical operations. A control group of men was investigated.

Therapy and survival in male breast carcinoma: A retrospective analysis of 50 cases.

The relationship between therapy and overall survival was retrospectively investigated in 50 patients with primary male breast carcinoma. Forty-five had radical or modified radical mastectomy and 5 simple mastectomy. Thirty-five received adjuvant post-operative therapy, including radiation, hormone and chemotherapy, given separately or in combination. The mean follow-up period was 67 (range, 1-230) months. The median survival was 33 months for patients receiving surgery alone and 86 months for those who also had adjuvant therapy (p=0.003). No difference in survival was found between simple or radical/modified radical mastectomy, nor among the various types of adjuvant therapy. Adjuvant therapy was most effective in large size, node positive and poorly differentiated tumors, and retained independent prognostic significance in multivariate analysis. With the limitation due to the small number of cases, our data suggest that adjuvant therapy may improve survival in males with cancer of the breast.

Oncogenes and male breast carcinoma: c-erbB-2 and p53 coexpression predicts a poor survival.

PURPOSE: To investigate the prognostic value of biomarkers in male breast carcinoma (MBC). PATIENTS AND METHODS: Fifty patients (mean age, 62.2 years) with invasive ductal carcinoma were retrospectively studied. All patients received surgery; 35 had adjuvant postoperative therapy. The median follow-up was 59 months (range, 1 to 230 months). c-myc, c-erbB-2, p53, and bcl-2 proteins were immunohistochemically detected on sections from formalin-fixed, paraffin-embedded tissues using 9E11, CB11, DO7, and bcl-2 124 monoclonal antibodies (mAbs). Estrogen, progesterone, and androgen receptors were detected using specific mAbs. Cell proliferation was assessed by MIB-1 mAb. RESULTS: In univariate analysis, c-myc, c-erbB-2, and p53 protein overexpression was significantly correlated with prognosis. The median survival was 107 months for c-myc-negative and 52 months for c-myc-positive patients (P =.01), 96 months for c-erbB-2-negative and 39 months for c-erbB-2-positive patients (P =.02), and 100 months for p53-negative and 33 months for p53-positive patients (P =.0008). Tumor histologic grade (P =.01), tumor size (P =.02), patient age at diagnosis (P =.03), and MIB-1 scores (P =.0004) also had prognostic value. In multivariate analysis, only c-erbB-2 and p53 immunoreactivity retained independent prognostic significance. All nine patients who did not express c-erbB-2 and p53 proteins were alive after 58 months, whereas none of the 14 patients expressing both proteins survived at 61 months follow-up (P =.0002). CONCLUSION: Overexpression of c-myc, c-erbB-2, and p53 proteins may be regarded as an additional prognostic factor in MBC. The combination of c-erbB-2 and p53 immunoreactivity can stratify patients into different risk groups.

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.

Androgen receptor expression in male breast carcinoma: lack of clinicopathological association.

Androgen receptor (AR) expression was retrospectively analysed in 47 primary male breast carcinomas (MBCs) using a monoclonal antibody on formalin-fixed, paraffin-embedded tissues. AR immunopositivity was detected in 16 out of 47 (34%) cases. No association was found with patient age, tumour stage, progesterone receptor (PGR) or p53 protein expression. Well-differentiated MBCs tended to be AR positive more often than poorly differentiated ones (P = 0.08). A negative association was found between ARs and cell proliferative activity: MIB-1 scores were higher (25.4%) in AR-negative than in AR-positive cases (21.11%; P = 0.04). A strong positive association (P = 0.0001) was found between ARs and oestrogen receptors (ERs). In univariate analysis, ARs (as well as ERs and PGRs) were not correlated with overall survival; tumour histological grade (P = 0.02), size (P = 0.01), p53 expression (P = 0.0008) and MIB-1 scores (P = 0.0003) had strong prognostic value. In multivariate survival analysis, only p53 expression (P = 0.002) and histological grade (P = 0.02) retained independent prognostic significance. In conclusion, the lack of association between AR and most clinicopathological features and survival, together with the absence of prognostic value for ER/PGR status, suggest that MBCs are biologically different from female breast carcinomas and make it questionable to use antihormonal therapy for patients with MBC.

Bcl-2 expression in male breast carcinoma.

We have analysed the expression of bcl-2 protein retrospectively in 34 primary male breast carcinomas (MBC), using the monoclonal antibody bcl-2 in formalin-fixed, paraffin-embedded tissues. Bcl-2 expression was compared with tumour clinicopathological features, sex steroid hormone receptors, DNA content, p53 immunoreactivity and cell proliferative activity assessed by counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against proliferating cell nuclear antigen and the monoclonal antibody MIB-1. Most (28, or 82.3%) of the 34 cases of MBC were bcl-2 positive. No association was found with clinicopathological features of the tumours, although bcl-2 tended to be more frequently expressed in small tumours (P=0.09) and in cases without necrotic areas (P=0.1). Nor was any association found with hormone receptor status, p53 immunoreactivity, DNA content, cell proliferative activity or patient survival. In multivariate analysis, only proliferative activity (expressed by AgNOR counts) and p53 immunoreactivity had independent prognostic significance. Our results indicate that MBC differs from FBC in that in MBC bcl-2 protein is not related to an oestrogen-dependent transcription pathway and bcl-2 alone is not sufficient to induce increased proliferation. These characteristics, together with the high prognostic value of cell proliferation and the lack of prognostic significance for hormone receptor status, support the hypothesis that MBC is biologically different from FBC.

Prognostic value of AgNOR counting.

The prognostic significance of the argyrophilic nucleolar organizer regions (AgNORs) in tumour pathology is still a matter of debate. A prospective study was performed in a series of renal cell carcinomas to clarify the prognostic value of AgNOR counting. Sections from 21 renal cell carcinomas were stained in 1990 with the method of Ploton. Black dots within the nucleus from 200 tumour cells were counted: the mean AgNOR count for the whole series was 6.13, the median 5.94 and the SD 1.78. Patients were then followed up for at least 6 years or to death: at the time of the survival analysis (June 1996), 13 patients were alive without evidence of recurrence or metastasis, 6 had died of the disease and 2 of myocardial infarction. All the patients with 5.94 AgNORs per cell or fewer were alive at 6-year follow-up, while only 60% of patients with more than 5.94 AgNORs per cell survived (p=0.01). In the multivariate analysis, only AgNOR count (p=0.015) retained an independent prognostic significance. With the limitation due to the small number of cases, this prospective study clearly indicates that AgNOR count has a significant prognostic role, at least in renal cell carcinoma.

DNA ploidy and p53 expression correlate with survival and cell proliferative activity in male breast carcinoma.

DNA flow cytometry and the monoclonal antibody DO7 were applied in formalin-fixed, paraffin-embedded specimens from 34 primary male breast carcinomas to verify whether DNA ploidy and p53 expression were associated with survival and proliferative activity. They were compared with tumor clinicopathologic features, sex steroid hormone receptors and cell proliferative activity, assessed by the counts of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody PC10 against the proliferating cell nuclear antigen and the monoclonal antibody MIB-1. A significant correlation was found between survival and tumor ploidy (median survival, 77 months for diploid but only 38 months for aneuploid cases; P = .03) and p53 expression (median survival, 95 months for cases with p53 scores < or = 14.06% versus 33 for cases with P53 scores > 14.06%; P = .0004; median survival, 99 months for p53 negative vs 39 for positive cases; P = .007). Tumor histological grade (P = .006), AgNOR counts (P = .0001), PC10 scores (P = .002), and MIB-1 scores (P = .001) were also associated with prognosis. In the multivariate analysis, only p53 scores (P = .001) or p53 immunopositivity (P = .003) and AgNOR counts (P = .022) retained an independent prognostic significance. Aneuploid tumors had higher AgNOR counts (P = .002), PC10 (P = .007), MIB-1 (P = .006), and p53 scores (P = .01) than diploid cases. A linear relationship was observed between p53 scores and AgNOR counts (r = .41; P = .014), PC10 (r = .46; P = .005), and MIB-1 scores (r = .44; P = .011). These results indicate that DNA ploidy and p53 expression are associated with survival and cell proliferative activity in male breast carcinoma. Quantitative parameters, such as DNA ploidy, p53 scores, AgNOR counts, PC10, and MIB-1 scores substantially improve the prognostic significance of the traditional parameters in male breast carcinoma.

Role of the argyrophilic nucleolar organizer regions in tumor detection and prognosis.

The expression of the argyrophilic nucleolar organizer regions (AgNORs) has been analyzed in renal, bladder, and pharyngeal carcinomas, multiple myeloma (MM), and skin melanocytic lesions to clarify their role in tumor detection and prognosis. Sections from formalin-fixed, paraffin-embedded biopsies were stained with the method of Ploton; the mean AgNOR number per nucleus (AgNOR count) and their distribution (configuration) were assessed examining 100 neoplastic cells. AgNOR counts and histologic grade were highly associated in bladder urotheliomas (6.01 for grade 1 [G1], 7.69 for G2, 13.35 for G3; p < 0.00001) and MM (3.18 for G1, 4.36 for G2, 6.13 for G3; p < 0.0001); they were not associated in renal cell carcinomas (5.35 for G1, 5.92 for G2, 7.99 for G3; p = 0.132) and pharyngeal carcinomas (11.1 for G2, 10.27 for G3; p = 0.08). AgNOR number was also related to the degree of malignancy in melanocytic lesions (2.93 for common blue nevus, 2.89 for benign nevus [BN], 3.69 for cellular blue nevus [CBN], 7.71 for malignant melanoma, and 8.33 for malignant cellular blue nevus [MCBN]; p < 0.00001). Association between AgNOR counts and pathologic stage was found in bladder carcinomas (6.43 for pTa, 10.19 for pT1, 12.57 for pT2-4; p < 0.00001) and MM (3.06 for cases with percentage of bone marrow plasma cells [BMPC%] < or = 20, 4.28 for BMPC% 21 to 50, 5.14 for BMPC% > 50; p < 0.0001]; no correlation was found in pharyngeal (11.18 for T1, 10.08 for T2, 10.68 for T3, 11.47 for T4; p = 0.18) or renal cell carcinomas (6.06 for pT2, 6.31 for pT3; p = 0.78). Few, large and grouped AgNORs were found in well-differentiated bladder carcinomas, MM, and benign melanocytic lesions; numerous, small and dispersed AgNORs were seen in poorly differentiated bladder, renal and pharyngeal carcinomas, MM and malignant melanocytic lesions. Significant association with prognosis was found in pharyngeal carcinomas (5-year survival: 68% for cases with < or = 10.31 AgNOR/cell, 20% for cases with > 10.31 AgNORs) and MM (5-year survival: 46% for cases with < or = 4.62 AgNOR/cell, 7% for cases with > 4.62 AgNORs; in MM the configuration too was related to prognosis: median of survival 72 months for tightly grouped, 16 for partially grouped, and 11 for dispersed AgNORs). Our results indicate that AgNOR number and configuration are useful in detection and prognosis of some neoplasias. They permit a rapid evaluation of morphology and tumor cell kinetics even on small biopsies.(ABSTRACT TRUNCATED AT 400 WORDS)

P53 overexpression correlates with proliferative activity and prognosis in carcinomas of the pyriform sinus.

p53 overexpression and proliferative activity were investigated in 28 squamous cell carcinomas of the pyriform sinus of the hypopharynx prior to therapy, using DO1 and MIB-1 monoclonal antibodies in routinely processed biopsies. MIB-1 scores were associated with tumour histological grade (35.4% for grade 3 versus 23.8% for grade 2 cases; p=0.008) and survival (the median of survival was 23 months for cases with MIB-1 scores less than or equal to 33.8% but 11 months only for cases with MIB-1 scores >33.8%; p<0.001). p53 scores were associated with tumour histological grade (56.5% for grade 3 versus 37.1% for grade 2 cases; p=0.02) and survival (median of survival 20 months for cases with p53 scores less than or equal to 56.2% versus 11 months for cases with p53 scores >56.2%; p=0.002). Tumour histological grade was also correlated with prognosis (median of survival 50 months for grade 2 versus 14 months for grade 3 cases; p=0.03). In the multivariate analysis, only MIB-1 (p=0.001) and p53 scores (p=0.003) had an independent prognostic significance. A linear relationship between p53 and MIB-1 scores was observed (r=0.54; p=0.012). With the limitation due to the small number of cases, our findings indicate that p53 overexpression correlates with proliferative activity and survival in squamous cell carcinomas of the pyriform sinus, and suggest the use of p53 and MIB-1 immunostainings in the pretherapeutic assessment of the tumour aggressiveness.

Proliferative activity is a significant prognostic factor in male breast carcinoma.

The proliferative activity of male breast carcinoma has been investigated using the staining of the argyrophilic nucleolar organizer regions (AgNORs), the monoclonal antibody against the proliferating cell nuclear antigen (PC10) and the monoclonal antibody MIB-1 in formalin-fixed, paraffin-embedded specimens from 27 primary male breast carcinomas at diagnosis. A significant correlation was found between survival and AgNOR counts (median of survival 77 months for cases with AgNOR/cell < or = 7.27 but 37 months only for cases with > 7.27 AgNOR/cell; P = 0.001), proliferating cell nuclear antigen scores (median of survival 73 months for cases with proliferating cell nuclear antigen < or = 18.25% versus 41 for cases with proliferating cell nuclear antigen > 18.25%; P = 0.013) and MIB-1 scores (median of survival 73 months for cases with MIB-1 scores < or = 23.5% versus 37 months for cases with MIB-1 scores > 23.5%; P = 0.01). Tumor histological grade was also correlated with prognosis (median of survival 72 months for grade 2 versus 33 months for grade 3 tumors; P = 0.01). Estrogen and progesterone receptors, immunohistochemically detected on paraffin-embedded sections, had no prognostic value. In the multivariate survival analysis, only AgNOR counts (P = 0.007) and tumor size (P = 0.003) had an independent prognostic significance. Our results indicate that methods for assessing the cell proliferation in routinely processed specimens offer significant prognostic information in male breast carcinoma. The finding, together with the lack of prognostic significance for estrogen receptors and progesterone receptors, suggests that male breast carcinoma is biologically different from female breast cancer.

Proliferative activity in the malignant cellular blue nevus.

The proliferative activity of four malignant cellular blue nevi (MCBN) was assessed in routinely fixed, paraffin-embedded material using staining for the argyrophilic nucleolar organizer regions (AgNORs), immunohistochemical staining for proliferating cell nuclear antigen (PCNA [PC10]), and DNA flow cytometry. The objective was to determine whether the evaluation of proliferative activity could represent a useful diagnostic parameter. Four cellular blue nevi (CBN), 10 melanocytic nevi (MN), four common blue nevi (BN), and 10 conventional malignant melanomas (MMs) were selected as controls. In the MCBN the mean AgNOR number, evaluated on the basis of 100 tumor cells, was 8.33 +/- 0.83; NORs were small and dispersed throughout the nucleus; the mean PCNA score was 31.93% +/- 4.4; and two of the cases were aneuploid and two diploid. In the CBN the AgNOR count was 3.69 +/- 0.56; NORs were large and mainly grouped in a central cluster; the mean PCNA score was 3.53% +/- 1.28; and three of the cases were diploid and one aneuploid. The AgNOR counts in the MCBN were significantly different from those in the CBN (P = .0002), MN (3.04; P = .00001), and BN (2.93; P = .00006), whereas they were not significantly different from those in the conventional MMs (7.64; P = .58). The PCNA (PC10) scores in the MCBN were significantly different from those in the CBN (P = .00003), MN (2.05%; P = .00001), and BN (5.06%; P = .00002), whereas they were not significantly different from those in the conventional MMs (28.9%; P = .49). In all the cases a linear relationship between AgNOR counts and PCNA scores was observed (r = .94, P = .00001). Our results indicate that AgNOR analysis and PCNA immunostaining can be regarded as useful additional parameters for the diagnosis of MCBN.

In situ hybridization for human papillomavirus as a method of predicting the evolution of cervical intraepithelial neoplasia.

Twenty-four women with cervical condylomata which were immunohistochemically positive for human papillomavirus (PV-Ag) (15 with CIN 1 and 9 with CIN 2) were followed for a period of 2-65 months. Fifty-seven biopsies were studied by the in situ hybridization (ISH) procedure for the detection of HPV 6/11 and 16/18 DNA. ISH positivity was found in 13/24 cases (54.2%); HPV 16/18 was evident in 7/9 CIN 2 (77.8%) as against 3/15 CIN 1 (20%) (P = 0.017) and in 8/13 cases with koilocytosis affecting up to 2/3 of the epithelial thickness (61.5%) as against 2/11 cases with koilocytosis affecting more than 2/3 of the epithelial layer (18.2%) (P = 0.03). Progression to CIN 3 occurred in 4 cases (2 CIN 1 and 2 CIN 2), the degree of dysplasia remained static in 5 cases (1 CIN 1 and 4 CIN 2) and regression occurred in 15 cases (9 CIN 1 and 6 CIN 2). The immunoperoxidase (IP) positive staining for PV-Ag persisted in 5/24 cases and disappeared in 19/24; 6/13 ISH positive cases maintained ISH positive and 7/13 became negative. The progression of dysplasia was significantly related to disappearance of the IP positivity (P less than 0.0001), to the ISH positivity (P = 0.05), to the persistence of ISH positivity (P = 0.008) and to HPV 16/18 positivity (P = 0.01). We believe that ISH positivity for HPV 16/18 in CIN 1 or 2 with low degrees of koilocytosis and conversion from PV-Ag positive to negative indicate a high risk of progression to CIN 3.

Argyrophilic nucleolar organizer region counts and Ki-67 scores in human renal cell carcinoma.

The proliferative activity of 21 cases of renal cell carcinoma has been investigated by means of monoclonal antibody Ki-67 and Nucleolar Organizer Regions (AgNORs) analysis. The correlation between AgNOR counts and Ki-67 scores was only slightly significant (r = 0.53, r2 = 0.28) as determined by linear regression. Positive correlation was found between Ki-67 scores and tumour histologic grade. However, no correlation was observed between Ki-67 scores and tumour pathologic stage and between AgNOR counts and tumour histologic grade and/or pathologic stage. The results suggest that AgNOR counts cannot replace Ki-67 scores in evaluating the proliferative activity of renal cell carcinoma and that such activity and both histologic grade and pathologic stage seem to be independent parametres.

Argyrophilic nucleolar organizer region counts in malignant melanoma associated with benign intradermal nevus.

A silver colloidal technique to demonstrate argyrophilic proteins of the nucleolar organizer regions (AgNORs) was performed on sections of 20 cases of malignant melanoma (MM) associated with underlying benign nevus (BN). In these cases, significant different AgNOR counts were found for MM and BN. In addition, this technique permitted the identification of melanocytic cells located between malignant and benign cells showing AgNOR scores intermediate (5.51) between BN (2.6) and MM (7.71) with a more complex and bizarre morphology than that observed in BN. The AgNOR technique can be suitable in the identification of residual nevus cells in MM, especially when their number is minimal and the common histologic criteria are unsatisfactory; it can also increase the understanding of the natural history of MM.