Novel biomarkers and genomic tests in prostate cancer: a critical analysis.

The aim of this review is to critically analyze the current state of research in selected biomarkers and genomic-based tests for prostate cancer (PCa) diagnosis, staging, prognostication, and monitoring. Although in Western societies, PCa is the most common solid malignancy and the second leading cause of cancer death in men, the vast majority of men with PCa are diagnosed with clinically localized disease. The widespread use of prostate-specific antigen (PSA) testing, on one hand, has resulted in earlier PCa detection at a potentially more curable stage, but on the other hand has led to an increase in the rate of negative biopsies, as well as overdetection and overtreatment of potentially indolent tumors that would not have become life-threatening to a patient. A multitude of molecular tests and algorithms has been developed to enhance diagnostic accuracy, improve pretreatment and post-treatment patient risk stratification, and identify aggressive versus indolent disease to facilitate therapeutic decision-making. PSA and derivatives (PSA kinetics, PSA density, percentage of free PSA) as well as algorithms based on PSA and PSA isoforms measurements (prostate health index, four-kallikrein score), urinary molecular biomarkers-based tests (Prostate Cancer Antigen 3, and the Michigan Health System Prostate Score) and selected genomic/proteomic tests now commercially available for disease prognostication (such as Confirm MDx, Prostate Core Mitomic Test, Oncotype DX, Prolaris, ProMark, and Decipher) are herein discussed to inform the readers about current and future clinical applications and their limitations. Finally, we briefly touch upon potential biomarkers predictive of response to therapy, such as androgen receptor splice variant AR-V7, and detection and quantification of circulating tumor cells in the blood stream.

Prediction of prostate cancer to urethra distance by a pretreatment nomogram: urethral thermoprotection implication in cryoablation.

INTRODUCTION: Cryoablation is a treatment option for prostate cancer (PCa) patients. A urethral warming catheter is placed to protect the prostatic urethra from cryo-injury. Thus tissue within certain depth beneath the urethral mucosa, including PCa in that zone, is not cryoablated. Preoperative predictors of PCa-to-urethra distance are important for urologists and patients to decide if undergoing cryoablation. METHODS: A total of 267 consecutive radical prostatectomy specimens were reviewed by a pathologist and the shortest PCa-to-urethra distance was recorded as 0 (PCa at urethra), 0.1-1 mm, 1.1-2 mm, 2.1-3 mm, 3.1-4 mm, 4.1-5 mm and >5 mm. Preoperative serum PSA (iPSA) and prostate biopsy (Bx) parameters such as highest Bx Gleason score (BxGS), number of positive cores, highest percentage of PCa/cores, bilateral disease, perineural invasion (PNI) and PCa location were also recorded. The PCa-to-urethra distance subdivided into two (3 and >3 mm) and all seven categories was correlated with iPSA and Bx parameters. Logistic and linear regression were used to analyze the data. RESULTS: Patients' median age and iPSA were 59 years and 5.28 ng ml(-1), respectively. PCa-to-urethra distance was <5 mm in 163 (61%) patients, 3 mm in 48% of patients. Significant univariate associations were found between shorter PCa-to-urethra distance and increasing iPSA (P<0.0001), BxGS (P=0.0016), number of positive cores (P< 0.0001), highest percentage of PCa/cores (P< 0.0001), bilateral disease (P=0.0003), PNI (P=0.01) and PCa detected in biopsies from apex (P< 0.0001), base (P=0.001) and base/medial base (P= 0.0006). In multivariate analysis, the iPSA (log), highest percentage of PCa/cores and PCa detected in the apex were significantly associated (P<0.0001) with both versions of PCa-to-urethra distance. CONCLUSIONS: Increasing iPSA, highest percentage of PCa/cores and PCa detected in the apex were associated with a shorter PCa-to-urethra distance. Inclusion of these preoperative parameters in a nomogram will help estimating the PCa-to-urethra distance and identifying better candidates for cryoablation.

Phase II trial of neoadjuvant nab-paclitaxel in high risk patients with prostate cancer undergoing radical prostatectomy.

PURPOSE: Taxane based chemotherapy has activity in advanced prostate cancer but previous studies of neoadjuvant docetaxel demonstrated a prostate specific antigen response with no obvious antitumor activity. The efficacy and safety of neoadjuvant albumin-bound paclitaxel (nab-paclitaxel, Abraxane), a novel nanoparticle based formulation, were assessed in patients with high risk, locally advanced prostate cancer. MATERIALS AND METHODS: Eligible patients had locally advanced prostatic adenocarcinoma, clinical stage cT2b or greater, Gleason score 8 or greater, or serum prostate specific antigen 15 ng/ml or greater without metastatic disease. Patients received 2 cycles of 150 mg/m(2) nab-paclitaxel weekly for 3 weeks during each 4-week cycle, followed by radical prostatectomy with bilateral lymphadenectomy. Efficacy assessments included pathological and prostate specific antigen response. RESULTS: A total of 19 patients completed neoadjuvant therapy and 18 underwent radical prostatectomy. Median pretreatment prostate specific antigen was 8.5 ng/ml and median Gleason score was 8. Despite the lack of complete pathological responses 5 of 18 patients (28%) had organ confined disease and 9 of 18 (50%) had specimen confined disease. Post-chemotherapy prostate specific antigen was decreased in 18 of 19 (95%) patients and median decrease was 2.9 ng/ml (35%, p <0.001). An initial prostate specific antigen after radical prostatectomy of 0.02 ng/ml or less was achieved in 17 of 18 (94%) patients. There were no significant perioperative complications. Cytoplasmic vacuolization (focal in 10 and extensive in 7) was evident in all but 1 patient (94%). Ten patients (56%) had grade 3 and 1 had grade 4 neutropenia with no febrile neutropenia. CONCLUSIONS: Neoadjuvant nab-paclitaxel was well tolerated. Similar to our experience with neoadjuvant docetaxel there were no pathological complete responses, although a possible histological antitumor effect was observed.

Parasagittal biopsies are more important as part of an initial biopsy strategy than as part of a repeat biopsy strategy: observations from a unique population.

Comparing the yield of parasagittal biopsies during initial saturation biopsy to the yield during repeat saturation biopsy for detection of prostate cancer. Office-based saturation biopsy (24 cores) with periprostatic lidocaine block was performed in 139 consecutive men who had never previously undergone prostate biopsy. Indication for biopsy was elevated prostate-specific antigen >2.5 ng/dl. Biopsy specimens were obtained and marked by location for histological examination. Subanalysis of patients from this unique study was performed to compare the location of saturation biopsy cancer detection in these patients to a cohort of 100 patients who had previously undergone biopsy with nonmalignant findings. In the initial biopsy group, cancer was detected in 62/139 patients (44.6%). Breakdown of cancer location demonstrated unique parasagittal cancers in 9/62 patients (14.5%). Laterally base cancer was found exclusively in 22/62 patients (35.5%). For the repeat biopsy population, cancer was found in 25 patients (25%); no patients (0%) had exclusive parasagittal cancer. To our knowledge, this is the first study to demonstrate a difference in the location of positive cores between initial and repeat biopsy status. The exclusive parasagittal cancer detection rate decreases significantly in the repeat biopsy population when using the same biopsy method. Our findings support including traditional template parasagittal sampling of the prostate on first-time biopsy in addition to lateral cores typical of extended field biopsies for a total of 10-12 cores. However, parasagittal sampling adds negligible additional information in repeat biopsy; thus we recommend obtaining primarily laterally based cores for repeat biopsy.

Acquisition of new "words" in normal subjects: a suggestion for the treatment of anomia.

The study explores the efficacy of three learning methods in normal controls. Thirty subjects, randomly assigned to the repetition, reading aloud, or orthographic cueing method, were asked to learn 30 new "words" (legal nonwords arbitrarily assigned to 30 different pictures); 30 further new "words" were used as controls. Number of trials to criterion was significantly lower, and number of words remembered at follow-up was significantly higher for the orthographic cueing method. Two aphasic patients with damage to the output lexicons were also rehabilitated with the same three methods. In both patients the orthographic cueing method was significantly more efficacious. The differences in learning efficacy of the three methods are discussed.

Demonstrability of the glycoprotein A-80 in postradiation prostatic carcinoma.

Radiation therapy results in significant morphological changes in prostatic carcinoma, including decreased cancer size, acinar shrinkage and distortion, cytoplasmic vacuolization, and nuclear pyknosis. Benign acini usually display enlarged, atypical cells with hyperchromatic nuclei. These changes confound the evaluation of limited postradiation samples. The glycoprotein A-80 is known to be upregulated in prostatic intraepithelial neoplasia (PIN) and prostatic carcinoma. In this study, we assessed the expression of A-80 in radiation-treated prostatic carcinoma. Paraffin sections from 64 postradiation prostatic carcinomas obtained at salvage prostatectomy were immunostained with a monoclonal antibody to A-80; selected sections were doubly immunostained with antibodies to A-80 and various cytokeratin polypeptides. All cases showed readily detectable and often intense staining in the cytoplasm of cancer cells and in intraluminal material of malignant acini. The extent and intensity of the reactions were independent of cancer size and grade. Strong reactions were seen in preserved and distorted acini, clear cell areas, single cancer cells, and in colloid pools with few or no recognizable cancer cells. PIN was present in 34 cases (53%), of which 27 (79%) stained strongly for A-80; atrophic and hyperplastic acini generally did not stain, irrespective of the degree of cellular atypia. The A-80 glycoprotein appears remarkably durable and is readily demonstrable in postradiation prostatic carcinoma despite profound architectural and cytologic changes. This characteristic may prove useful in evaluating small samples for confirmation of diagnosis and determination of extent of residual or recurrent prostatic carcinoma after radiation therapy.

Proliferation, apoptosis and cell cycle regulation in prostatic carcinogenesis.

Cancer progression occurs because of imbalance in the processes of proliferation, differentiation and programmed cell death. In prostate cells, these processes are regulated at least in part by androgens. Structural alterations occur in a variety of genes regulating such processes. In order to obtain meaningful information on the biologic behavior of prostate cancers, it is important to assess androgen dependence and alterations in key genes regulating cell cycle kinetics as well as the aberrant response in cellular proliferation and death that such genetic events bring about. Most genetic alterations resulting in cancer progression alter normal cell cycle progression. Assessment of cell division cycle alterations by means of quantitative methods may have prognostic value, while interference with cell cycle regulatory proteins may result in powerful therapeutic tools. Here we review the methodologies utilized in the assessment of cell cycle kinetics and the abnormalities in proliferation and apoptosis encountered in prostate cancer. In addition, alterations in novel, important genes likely to have an impact on the behavior of prostate cancer and its precursor lesions are discussed. Molecular assessment of genetic alterations in genes that play a pivotal role in prostate cancer coupled with quantitative cytometry of cell kinetics may be utilized for a more precise evaluation of biologic behavior of prostate neoplasms.

Automated in situ hybridization: diagnostic and research applications.

Although in situ hybridization has been in use for almost 30 years, its technically demanding nature, the requirements for optimal tissue fixation and preservation, and the turnaround time for the experiments have prevented this technique from becoming widely used in the surgical pathology setting. The use of nonisotopic reporter molecules, the possibility of performing hybridization on archival material, and very recently, automation of the procedure have brought in situ hybridization to the forefront of diagnostic and experimental pathology. We describe our experience with nonradioactive, automated in situ hybridization, compare the technique with traditional manual procedures, and briefly outline its potential applications in diagnostic pathology and in the research setting.

Mitogen-activated protein kinases and apoptosis in PIN.

Mitogen-activated protein (MAP) kinases are key elements of the signalling systems needed to transduce different extracellular messages into cellular responses. At least three parallel MAP kinase pathways have been identified: one, stimulated by serum and growth factors to activate extracellular signal-regulated protein kinases (ERKs) by dual tyrosine and threonine phosphorylation, triggers cell proliferation or differentiation; the other two, induced by a variety of cellular stresses to activate c-jun N-terminal kinases (JNKs) and reactivating kinase (p38/RK), result in growth arrest and induction of apoptosis. Mitogen-activated protein kinase phosphatases (MKPs) inactivate MAP kinases through dephosphorylation and, thus, can modulate the MAP kinase pathways. Expression of JNK-1, ERK-1, p38/RK and MKP-1 proteins was investigated by immunohistochemistry and expression of MKP-1 mRNA by in situ hybridisation in 50 cases of high-grade prostatic intraepithelial neoplasia (PIN), thought to represent the precursor of prostate cancer. The frequency of apoptotic cells was also determined in these cases. Overexpression of the three MAP kinases and MKP-1 mRNA was found in all cases of high-grade PIN compared with normal prostate. Immunoreactivity for MKP-1 protein was found to be as intense as in normal glands in 30% and weaker in 56% of the PIN cases. Fourteen per cent of PIN cases did not stain with MKP-1 antibody. The proportion of apoptosis was significantly higher (P < 0.008) in PIN lesions that did not express MKP-1 protein than in those that did. These results are consistent with our previous demonstration of preferential inhibition of the apoptosis-related kinases by MKP-1 and further support the contention that MKP-1, even in PIN, may shift the balance existing between cell proliferation and death. When expressed, it may inhibiting those pathways that lead to apoptosis.

Apoptosis in prostatic adenocarcinoma following complete androgen ablation.

OBJECTIVE: To analyze the cell death phenomenon in prostate cancer following complete androgen ablation. METHODS: The frequency and location of apoptotic bodies (ABs) were evaluated in haematoxylin and eosin stained sections of radical prostatectomy specimens from patients with invasive prostatic adenocarcinoma treated with neo-adjuvant endocrine combination therapy for 3 months before surgery. The results were compared with an untreated age- and stage-matched control group. RESULTS: Both in treated and untreated prostate tissue the AB frequency increased from normal prostate, through prostatic intraepithelial neoplasia, up to prostatic adenocarcinoma. The main location was in the cell layers adjacent to the stroma, their frequency decreasing towards the lumen. The frequency of ABs was higher in the treated prostate glands than in the untreated groups. The relative increase of the AB frequency in treated carcinomas as compared with untreated ones was lowest in tumours with a solid pattern, intermediate in the cribriform, and highest in the acinar pattern. CONCLUSIONS: Complete androgen ablation induces involution of prostate tissue mainly through the enhancement of apoptosis. This type of cell death is thought to play a major role and might be linked to specific changes in signal transduction mechanisms in response to hormonal withdrawal.

Heterogeneity of androgen receptor content in advanced prostate cancer.

The principal mode of treatment of advanced (late stage) prostate cancer is androgen ablation. Although the response rate to hormonal ablation is high, relapse ultimately leading to death occurs in the majority of patients in remission from outgrowth of androgen-independent tumor cells. High-grade and high-stage cancers are more likely to progress to androgen independence. This study was undertaken to analyze the expression level of androgen receptor (AR) protein in prostatic carcinomas in relationship to grade and stage of disease. AR protein expression was assessed in 40 archival cases of prostate carcinoma by automated immunohistochemical techniques with standardized development times. Positive nuclei were quantitated by computer-assisted image analysis. Eighty-five percent of the prostatic carcinomas showed high levels of expression, defined as having AR present in more than 50% of the cells by light microscopy. Results of image analysis demonstrated that the variability of AR protein content per unit nuclear area increased with increasing grade (P < .03), regardless of cell size. High-grade prostatic intraepithelial neoplasia (PIN), present in 17 (42.5%) of the 40 cases, showed markedly reduced AR nuclear staining, compared with low-grade PIN or normal prostate. We show that AR content in prostate tumor cells becomes more variable with increasing Gleason score. In high-grade PIN, the in situ precursor of invasive prostate cancer, AR expression is either downregulated and/or restricted to the cytoplasm, but it is not heterogeneous. These data suggest that the heterogeneity in the expression of the receptor increases with progression of invasive prostate cancer and might in part account for a variable response to endocrine therapy.

The cell cycle inhibitor p27 is an independent prognostic marker in small (T1a,b) invasive breast carcinomas.

Breast carcinomas < or = 1 cm in size (T1a,b) are being detected more frequently as a result of screening. Because traditional prognostic parameters are either lacking (tumor size) or rare (nodal metastases), a marker(s) is needed to identify the subset of patients who could benefit from adjuvant therapy. A retrospective series of 202 patients with stage T1a,b invasive breast carcinomas was evaluated. The clinicopathological features (age, histological grade, extensive in situ carcinoma, hormone receptor status, and nodal metastasis) as well as microvessel density and the expression of c-erb-B2, p53, MIB-1/Ki-67, and cdc25B were assessed. In addition, expression of the cell cycle inhibitor p27 was evaluated. Nineteen patients (18% of patients who had axillary dissection) had locoregional lymph node metastases. Forty-two % of them died of disease (median survival, 112 months), whereas mortality was 11% in node-negative patients (median survival, 168 months; P = 0.0055). Patients with low p27 expression had a median survival of 139 months (17% mortality) versus 174 months (9% mortality) in the group with high p27 expression (P = 0.0233). Lack of p27 was associated with poor prognosis when node-positive patients were excluded (P = 0.0252). Nodal status and low p27 were found to be the only independent prognostic parameters by both univariate and multivariate analysis, with relative risks of dying of disease of 4.9 (P = 0.001) and 3.4 (P = 0.0306), respectively. Assessment of p27, which yields prognostic information in node-negative patients, could be useful to identify patients with small, invasive breast carcinomas who might benefit from adjuvant therapy.

Mitogen-activated protein kinase phosphatase 1 is overexpressed in prostate cancers and is inversely related to apoptosis.

Several oncogenes involved in prostate carcinogenesis activate mitogen-activated protein (MAP) kinases, which can relay both proliferative (via extracellular regulated kinases (ERK)) and apoptotic signals (via jun N-terminal protein kinases (JNK)) to the nucleus. Mitogen-activated protein kinase phosphatase 1 (MKP-1) is induced by several oncogenes in the ras-dependent pathway and can inactivate both MAP kinase pathways. The role of MKP-1 in proliferation and apoptosis is, however, still controversial. A series of 51 prostate cancers, including a subset (n = 13) that had been previously treated by androgen ablation, was used to examine whether MKP-1 mRNA and protein expression correlated with that of ERK-1, JNK-1, bcl-2, which confers resistance to apoptosis, and apoptotic index measured by in situ end-labeling of fragmented DNA. In a subset of tumors, MKP-1 expression was assessed by semiquantitative RT-PCR and was compared with both ERK-1 and JNK-1 enzymatic activity. In cases not treated by androgen ablation, MKP-1 was overexpressed in the preinvasive stage of prostate cancer, but its expression decreased with higher histologic grade and advanced disease stage. There was coexpression of MKP-1, ERK-1, and JNK-1 proteins. In addition, MKP-1 expression was inversely correlated to JNK-1 but not to ERK-1 enzymatic activity. Finally, MKP-1 and bcl-2 were inversely related to apoptotic indices. In cases treated by total androgen ablation, MKP-1 and bcl-2 were both down-regulated, whereas JNK-1 was up-regulated. Subpopulations of cells that did not undergo apoptosis maintained expression of both MKP-1 and bcl-2. These results suggest that MKP-1 overexpression is associated with the early phases of neoplastic transformation in prostate tissue. The enzymatic data on MKP-1 kinase substrates and the inverse correlation between MKP-1 and parameters of programmed cell death support the hypothesis that MKP-1 inhibits apoptosis in human prostate tumors, perhaps through the JNK pathway.

Expression of mitogen-activated protein kinase phosphatase-1 in the early phases of human epithelial carcinogenesis.

Many mitogens and human oncogenes activate extracellular regulated kinases (ERKs), which in turn convey proliferation signals. ERKs or mitogen-activated protein (MAP) kinases are inactivated in vitro by MAP kinase phosphatases (MKPs). The gene encoding one of these MKPs, MKP-1, is a serum-inducible gene and is transcriptionally activated by mitogenic signals in cultured cells. As MKP-1 has been shown to block DNA synthesis by inhibiting ERKs when expressed at elevated levels in cultured cells, it has been suggested that it may act as a tumor suppressor. MKP-1 mRNA and MAP kinase (ERK-1 and -2) protein expression was assessed in 164 human epithelial tumors of diverse tissue origin by in situ hybridization and immunohistochemistry. MKP-1 was overexpressed in the early phases of prostate, colon, and bladder carcinogenesis, with progressive loss of expression with higher histological grade and in metastases. In contrast, breast carcinomas showed significant MKP-1 expression even when poorly differentiated or in late stages of the disease. MKP-1, ERK-1, and ERK-2 were co-expressed in most tumors examined. In a subset of 15 tumors, ERK-1 enzymatic activity as well as structural alterations that might be responsible for loss of function of MKP-1 during tumor progression, were examined. ERK-1 enzymatic activity was found to be elevated despite MKP-1 overexpression. No loss of 5q35-ter (containing the MKP-1 locus) was detected by polymerase chain reaction in metastases compared with primary tumors. Finally, no mutations were found in the catalytic domain of MKP-1. These data indicate that MKP-1 is an early marker for a wide range of human epithelial tumors and suggest that MKP-1 does not behave as a tumor suppressor in epithelial tumors.

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.

Effect of microwave oven heating times on androgen receptor antigen retrieval from paraffin-embedded prostatic adenocarcinoma.

The aim of this study was to investigate the effect of microwave oven heating for antigen retrieval on the immunoreactivity of human prostate carcinoma androgen receptor (AR) in tissue sections. Formalin-fixed, paraffin-embedded tissue sections were microwaved at 5-min intervals for a total of 15, 20, 25, 30, 35 minutes at maximum power (700W). The monoclonal antibody F39.4.1 directed against human AR was used at a 1:10 dilution. Without microwave oven heating, prostatic tissue did not exhibit any AR immunoreactivity. Moderate positivity appeared after three 5-minute cycles of microwave heating. The intensity of immunoreactivity improved progressively with heating times of 20 and 25 min up to an optimum time of 30 minutes, when nuclear staining was most intense with the absence of background staining and without loss of morphological details. While antigen retrieval is effective in restoring antigenicity in a variety of setting, the length of time prostate tissue is exposed to microwave radiation is critical in order to obtain optimal AR immunostaining. AR immunostaining reliably permitted evaluation of the distribution and intensity of positively stained nuclei and the distinction of the various cell types in archival material.

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.

Molecular events in the early phases of prostate carcinogenesis.

Basal cells lining prostatic acini have unique morphologic and immunophenotypic characteristics. The role of these uncommitted cells in the genesis of cancer in the prostate is intriguing. Here, we discuss immunophenotypic and molecular features of basal cells of prostatic acini, and compare them with those of cytologically transformed cells of prostatic intraepithelial neoplasia (PIN) in both human tissues and animal models. Following a summary of the current concepts of molecular events in prostatic cancer, we will discuss the role of the ras-dependent pathway in early prostate carcinogenesis with a special emphasis on mitogen-activated protein kinase phosphatase (MKP-1). We will also outline the importance of techniques such as differential display-polymerase chain reaction (ddPCR) followed by in situ hybridization in the characterization of genes which may have a critical role in early prostate carcinogenesis. Finally, we will underscore the role of animal models in understanding the early events leading to neoplastic transformation of prostate cells.

Apoptotic bodies in prostatic intraepithelial neoplasia and prostatic adenocarcinoma following total androgen ablation.

The frequency and location of apoptotic bodies (AB) were evaluated in hematoxylin- and eosin-stained sections of 12 radical prostatectomies from patients with prostatic adenocarcinoma pre-treated for 3 months with total androgen ablation. Results were compared with an untreated age-, morphology- and stage-matched control group. Treated prostates showed involutional changes, with cytoplasmic vacuolization and chromatin changes ranging from mild to severe condensation, similar to that observed in apoptosis. In treated benign prostatic epithelium, the mean number of AB was 1.64% (standard error (SE), 0.19%), 6.3 times greater than in the untreated group (mean, 0.26%; SE, 0.03%). AB were more frequent in the basal cell layer than in the lumenal cell layer, whereas in the untreated group, AB were almost exclusively found in the basal cell layer. In treated prostatic intraepithelial neoplasia (PIN present in 10 cases), the mean number of AB was 1.74% (SE, 0.04%), which was 2.56 and 2.32 times greater than in untreated low grade PIN (mean, 0.68%; SE, 0.15%) and high grade PIN (mean, 0.75%; SE, 0.11%), respectively. In treated and untreated PIN, the number of AB was greatest in the basal cell layer, less in the intermediate cell layer and lowest in the cell layer bordering the lumen. The mean number of AB in the 12 treated cancers was 1.35 times greater than in untreated cancers (1.80% [SE, 0.12%] versus 1.33% [SE, 0.32%], respectively). The number of AB in treated cases of the acinar pattern of cancer (present in 7 cancer cases) was 1.78 times greater than in untreated cases, and in treated cases of the cribriform pattern of cancer (present in four cases), the mean number of AB was 1.39 times greater than in untreated cases. The number of AB was greatest in the outermost cell layer, with progressive decrease in layers closer to the lumen. In the one treated case with solid/trabecular pattern of cancer, the number of AB was 1.08 times greater than in untreated cases. The gland lumina was rich in macrophages, sloughed secretory cells with degenerative features and AB. The number of AB in the lumina increased from normal epithelium through PIN to cancer, and was greater in treated cases than in untreated cases.

Quantitative evaluation of nucleolar features on cytologic material in brain tumor diagnosis.

Nucleolar-related features were quantified in toluidin blue-stained smears from 36 brain tumors in order to improve our knowledge of the nucleolar frequency, size and margination. It was observed that low-grade astrocytic tumors had high percentages of nucleolated cells but the nucleoli were mostly single with maximum nucleolar diameter smaller than 2.00 microns. The percentages of marginated nucleoli were also low, ranging between 3.00% and 30.00% (only one case had a higher percentage). The high-grade tumors, i.e. anaplastic astrocytomas and glioblastomas, did not significantly differ from low-grade astrocytomas in their percentages of nucleolated nuclei, but they showed a higher number of nuclei having three or more nucleoli and the mean nucleolar diameter was in general bigger than 2.00 microns. Glioblastomas had marginated nucleoli much more frequently than anaplastic astrocytomas, the percentage in all but one case being higher than 30.00%. The percentage of marginated nucleoli was much higher in glioblastomas than in metastases, while the nucleoli were bigger in the latter group. A wide range of values for most of the nucleolar-associated parameters was observed in the remaining non-astrocytic brain tumors. Our results, showing differences in nuclear number, size and margination in different brain tumors, lead us to consider it worthwhile to investigate nucleolar-related features and their relationships using a quantitative approach.