Mitogen-activated protein kinase and mitogen-activated kinase phosphatase-1 expression in the Noble rat model of sex hormone-induced prostatic dysplasia and carcinoma.

Our recent studies have implicated the TGF-alpha/epidermal growth factor receptor pathway in the genesis of testosterone (T) and estradiol-17 beta (E2)-induced dysplasia in the dorsolateral prostate (DLP) of Noble rats. This pathway was also found to be markedly up-regulated in the androgen-independent transplantable carcinoma that arose from the DLP of a Noble rat. In the current study, we investigated the expression of mitogen-activated protein kinase (MAP-kinase) and mitogen-activated kinase phosphatase-1 (MKP-1), key downstream regulators of growth factor-activated signal transduction in the DLP of castrated, castrated T-supplemented, and T+E2-treated rats and in the androgen-independent transplantable carcinoma. Both MAP-kinase and MKP-1 expression in the DLP were found to be dependent on androgen stimulation. Immunoblots of DLP from T+E2 treated rats demonstrated a selective decline in MKP-1 levels with no alteration in MAP-kinase expression. These findings suggest that the dual hormone treatment induces changes in the signal transduction pathway, which favors the protracted mitogenic action of MAP-kinase. In situ hybridization and immunohistochemistry findings corroborated the immunoblot data but also revealed that both MAP-kinase and MKP-1 were strongly expressed in severely dysplastic lesions, which may indicate the presence of transformed cells in these foci. In this regard, both proteins were strongly expressed in samples of the androgen-independent transplantable carcinoma. Taken together, results from this and our recent study suggest that alterations in a growth factor-MAP-kinase pathway may be important events in the initiation and progression of prostatic carcinoma.

Immunohistochemical evaluation of type IV collagenase (72-kd metalloproteinase) in prostatic intraepithelial neoplasia.

The aim of this study was to investigate the expression of type IV collagenase (72-kd metalloproteinase, MMP-2) in prostatic intraepithelial neoplasia (PIN) in relation to normal prostate (NP) and prostatic adenocarcinoma (PAc). Twenty formalin-fixed, paraffin-embedded prostatectomy specimens, in which NP, PIN and PAc were present, were immunohistochemically examined. The NP ducts and acini not contiguous with PIN and PAc showed slight MMP-2 immunostaining in the secretory cells, with some increase in intensity at the apical border, and moderate to strong immunoreactivity of some basal cells. In NP adjacent to PIN and PAc, rare ducts and acini showed strongly stained cells either isolated or in small groups of two, located within the thickness of the epithelium, close to the basement membrane. In the majority of PIN ducts and acini, the stratified secretory cells showed moderate staining. Most of these ducts and acini also showed strongly stained cells, which were mostly isolated, and either in contact with the basement membrane or scattered among the secretory cells. Low and high grade PIN showed some difference in the frequency of dark cells, which were more numerous in the latter. A small group of neoplastic acini adjacent to high grade PIN (early invasive adenocarcinoma) was observed in one of the 20 cases. Intense immunostaining was present in the acini originating from the PIN lesion. MMP-2 immunostaining of PAc was heterogeneous in intensity and location. Cribriform and solid/trabecular PAc showed weak cytoplasmic immunostaining; both moderately and intensely stained cells were seen in the cell layer adjacent to the stroma, intense immunostaining was shown by small clusters of neoplastic cells or single neoplastic cells located in the stroma. In acinar PAc, weak cytoplasmic immunostaining for MMP-2 was seen throughout most areas of the tumours, whereas moderately and intensely stained cells were observed less frequently than in cribriform and solid/trabecular adenocarcinoma. Intense immunostaining of single or small clusters of neoplastic cells located in the stroma was occasionally observed and, as with cribriform and solid/trabecular PAc, mainly located towards the periphery of the tumour nodules. Occasional ducts and acini with PIN and foci of PAc were either completely negative or very weakly stained. In conclusion, MMP-2 immunostaining increases progressively from NP, through PIN, up to invasive PAc. These results directly support the hypothesis that increased expression of metalloproteinases is a marker of malignant conversion.

Morphometrically assisted grading of astrocytomas.

Alcohol-fixed, toluidine-blue-stained smears from 24 astrocytomas (12 low grade and 12 high grade or anaplastic) were included in the study. In each case 50 nuclei from representative areas of the tumor were selected for analysis; quantitative features pertaining to both the entire nucleus and the nucleoli were computed. Nuclear features were nuclear area and total optical density. Nucleolar features were number of nucleoli per nucleus, nucleolar area, variance of the nucleolar area, and mean and variance of the distance of nucleoli from the nuclear membrane. The results showed distinct changes in a number of nuclear and nucleolar features from low to high grade astrocytomas. Features expressing the most pronounced nuclear changes were area, grey level nonuniformity and run percentage. Changes were also found in the following nucleolar features: number of nucleoli, nucleolar area, variance of nucleolar area, nucleolar location and variance of nucleolar location. Linear discriminant analysis was carried out to determine a direction in feature space along which astrocytomas of low and high grade might be ranked. The nuclear area, number of low gray value pixels and a run length feature provided a useful linear combination. The study showed that one can derive a set of objective criteria from morphometric measurements that allows an ordering of astrocytoma cases along an axis and that might be used for continuous grading.

Quantitative characterization of the frequency and location of cell proliferation and death in prostate pathology.

This paper evaluates the use of quantitative methods to accurately assess cell proliferation and death in untreated and treated prostate lesions. The analysis of proliferating cell nuclear antigen (PCNA)-stained nuclei allow precise evaluation of the proliferating cells and exact identification of their location in the progression of untreated prostatic intraepithelial neoplasia (PIN) to prostatic adenocarcinoma (PAC). The evaluation of the frequency and location of apoptotic bodies (ABs) gives accurate information on the apoptotic phenomenon in PIN compared to normal prostate (NP) and PAC. In fact, the frequency of ABs increases from NP to PIN to PAC and parallels that observed with PCNA. However, the AB-related values were approximately one-eighth to one-tenth of those obtained with PCNA immunostaining. Combination endocrine therapy (CET) decreases the proliferative activity and enhances the apoptosis phenomenon in NP, PIN, and PAC. This might indicate that CET could induce a certain degree of regression not only of PAC, but also of PIN.

Frequency and location of mitoses in prostatic intraepithelial neoplasia (PIN).

The aim of our study was to assess the frequency and location of mitoses in routine haematoxylin- and eosin-stained sections of prostatic intraepithelial neoplasia (PIN) and then to compare the patterns with those in benign prostatic hyperplasia (BPH) and prostatic invasive adenocarcinoma (PAC). The frequency of mitoses in the epithelial cell layers increased from BPH through PIN up to PAC. The proportions of mitoses in PIN lesions of low grade (PINlow) and high grade (PINhigh) were greater than in BPH (mean 0.001%; standard error, SE, 0.001%), the values decreasing from the basal layer towards the lumenal. In PINlow, the mean category values were 0.087% (SE 0.04%) in the basal, 0.046% (SE 0.033%) in the intermediate and 0.024% (SE 0.024%) in the lumenal position. In PINhigh, the mean category values were 0.194% (SE 0.178%) in the basal position, 0.075% (SE 0.06%) in the intermediate and 0.049% (SE 0.033%) in the lumenal position. The proportions of mitoses in adenocarcinoma with cribriform pattern decreased from the basal towards the lumenal layer, as for PIN: 0.154% (SE 0.096%) in the basal position, 0.072% (SE 0.044%) in the intermediate and 0.064% (SE 0.04%) in the lumenal position. In the solid/trabecular adenocarcinomas, the mean category value in the cell layer adjacent to the stroma was 0.22% (SE 0.111%), whereas in the other cell layers it was 0.074% (SE 0.045). In small and large acinar adenocarcinomas, the proportions of mitoses were 0.058% (SE 0.024%) and 0.068% (SE 0.019%), respectively. In conclusion, the evaluation of mitotic frequency and location in haematoxylin- and eosin-stained sections gives accurate information on how the mitotic activity in PIN compares with BPH and PAC.

Prostatic intra-epithelial neoplasia. Qualitative and quantitative analyses of the blood capillary architecture on thin tissue sections.

The aim of our study was to qualitatively and quantitatively investigate the capillary architecture on lectin Ulex Europaeus agglutinin I-stained histological section in prostatic intra-epithelial neoplasia. The capillaries appeared as small, short or elongated vessels with either a smooth or undulated external contour and either virtual or visible lumen, sometimes with a clearly identifiable endothelial nucleus/i. In the benign prostatic hyperplasia and prostatic intra-epithelial neoplasia categories, the capillaries appeared located in close contact with (i.e. touching) or in proximity to the basement membrane of ducts and acini. In the invasive adenocarcinoma category, on the contrary, the capillaries in general appeared interspersed within the tumour stroma and septa. Our quantitative studies of the capillary architecture showed that, going from benign prostatic hyperplasia through prostatic intra-epithelial neoplasia up to invasive adenocarcinoma, an increasing proportion of capillaries becomes shorter, with open lumen and undulated external contour and with a greater number of endothelial cells. The highest proportion of touching capillaries was seen in benign prostatic hyperplasia, while the lowest was in invasive adenocarcinoma, being intermediate in prostatic intra-epithelial neoplasia. When the prostatic intra-epithelial neoplasia samples were divided into low-grade and high-grade, the feature values in the low-grade approached those in benign prostatic hyperplasia, whereas in the high-grade they were close to invasive adenocarcinoma. Half of the benign prostatic hyperplasia samples were taken from total prostatectomies because of the preoperative diagnosis of prostatic adenocarcinoma. The feature values in this subcategory were close to those of prostatic intra-epithelial neoplasia of low grade.

Proliferating cell nuclear antigen (PCNA) in prostatic invasive adenocarcinoma. Is the proliferation state in the marginal zone of the tumour higher than in the central part?

Expression and location of Proliferating Cell Nuclear Antigen in epithelial nuclei were assessed in invasive adenocarcinoma of the prostate gland. The PCNA-positive nuclei showed homogeneous or granular types of immunostaining or a mixture of both, and a gradation in the intensity of staining. Nuclei with homogeneous pattern appeared darker brown than the lighter granular and mixed patterns. Darker nuclei were quite frequently noted, mainly among the epithelial cells adjacent to the stroma. For the marginal zone of invasive adenocarcinoma, the mean proportion of PCNA-stained nuclei in the small acinar pattern was somewhat similar to that in the large acinar pattern, i.e., 8.66% and 9.06%, respectively. In contrast, the mean values in the cribriform pattern were greater than in the small and large acinar patterns, and decreased from the nuclei in the basal position, or adjacent to the stroma, toward the lumen: 14.40% in the basal position, 11.84% in the intermediate and 9.26% in the lumenal. In the solid/trabecular pattern, the proportions of PCNA-positive nuclei were higher than in all the other patterns: 17.60% in the cell layer adjacent to the stroma and 13.88% in the other layers. The trend of value changes in the central zone of the tumour was similar to that obtained in the marginal zone. However, the proportions were lower and the differences statistically significant. This might indicate that the proliferation state is higher in the marginal zone and that the tumour grows eccentrically rather than centrally.

Prostatic intra-epithelial neoplasia: expression and location of proliferating cell nuclear antigen in epithelial, endothelial and stromal nuclei.

The expression and location of proliferating cell nuclear antigen (PCNA) immunostaining in epithelial, endothelial and stromal nuclei were assessed in prostatic intra-epithelial neoplasia (PIN). It was then compared with patterns in benign lesions and in invasive adenocarcinomas of the prostate. The PCNA-positive nuclei showed homogeneous or granular types of staining, or a mixture of both, and a gradation in the intensity of staining. Nuclei with granular and mixed patterns appeared lighter brown than those with a homogeneous pattern, which are darker and more often noted in PIN and invasive adenocarcinomas than in benign lesions. For epithelial PCNA-stained nuclei, the proportions in the two grades of PIN were greater than in benign prostatic hyperplasia (mean 3.16%, SE 0.31%) and prostatic atrophic ducts and acini (mean 0.56%, SE 0.09%), the values decreasing from the nuclei in the basal position towards those in the luminal layer. In grade 1, the category mean values were 9.51% (SE 1.14%) in the basal, 7.02% (SE 1.27%) in the intermediate and 6.02% (SE 0.90%) in the luminal position. In grade 2, the category mean values were 13.81% (SE 1.42%) in the basal position, 10.99% (SE 1.17%) in the intermediate and 7.91% (SE 1.43%) in the luminal position. In small and large acinar adenocarcinomas, the proportions of positive nuclei were 8.66% (SE 0.30%) and 9.06% (SE 0.30%), respectively. The category mean values in the cribriform adenocarcinomas were 14.40% (SE 0.61%) in the basal position, 11.84% (SE 1.30%) in the intermediate and 9.26% (SE 0.66%) in the luminal position. As in PIN, the proportions of immunostained nuclei in the adenocarcinoma with cribriform pattern decreased from the basal towards the luminal layer. In the solid/trabecular adenocarcinomas, the category mean value in the cell layer adjacent to the stroma was 17.60% (SE 2.92%), whereas in the other cell layers it was lower than that in the cells adjacent the stroma (mean 13.88%, SE 1.71%). For capillary endothelial and stromal cells, the percentages of PCNA-stained nuclei were much lower than those in the epithelial component. The lowest mean values were obtained in benign lesions, whereas the highest were in invasive adenocarcinomas, the percentages in PIN being intermediate.

Aneuploidy and nuclear features of prostatic intraepithelial neoplasia (PIN).

Quantitative analyses (QAs) of prostatic intraepithelial neoplasia (PIN) helped to objectively define some traditional features of the lesion because, first, changes in value are represented by numbers and not by subjective evaluation of morphologic clues. QAs have also helped to identify subtle abnormalities. For example, the degree of nucleolar margination is a new diagnostic feature which can be easily evaluated as the proportion of nucleoli touching the nuclear membrane. Thirdly, QAs have provided useful insights into understanding some morphologic changes. PIN, in fact, appears to be characterized by complex changes which involve the secretory cells as well as the basal layer and which affect the surrounding stroma. In the epithelial lining, two types of simultaneous changes take place, the first in the nucleus (expression of abnormal proliferative and/or renewal activity) and the second in the cytoplasm (expression of the disorder in cell differentiation), pointing out that only PIN samples of high grade can be considered as having acquired the characteristics of a neoplastic lesion.