Protection against 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine cytotoxicity and DNA adduct formation in human prostate by glutathione S-transferase P1.

The prostate has been identified as a target for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced carcinogenesis. Humans are exposed to PhIP through ingestion of well-done cooked meats, and there is evidence from epidemiological studies that implicates red meat consumption in prostate carcinogenesis. The alpha and pi class isoforms of glutathione S-transferases (GSTs) have been shown to inhibit adduction of activated PhIP metabolites to DNA in cell-free systems. In humans, silencing of GST pi(GSTP1) through CpG island hypermethylation is found in nearly all prostate carcinomas and is believed to be an early event in prostate carcinogenesis. We hypothesized that suppressed GSTP1 expression in prostate cells would increase their vulnerability to cytotoxicity and DNA adduct formation mediated by activated PhIP metabolites. To test this hypothesis, the human prostate adenocarcinoma cell line, LNCaP, which contains a silenced GSTP1 gene, was genetically modified to constitutively express high levels of GSTP1. Both LNCaP and LNCaP-GSTP1 cells exposed to N-OH-PhIP, but not parent PhIP, for 24 h showed a dose-dependent decrease in cell viability. GSTP1-overexpressing cells had LC50s 30-40% higher than cells transfected with the vector alone. PhIP-DNA adducts isolated from LNCaP-derived cells and primary human prostate tissue cultures exposed to N-OH-PhIP were analyzed by liquid chromatography/electrospray ionization mass spectrometry. Primary cultures of human prostate tissue and LNCaP-GSTP1 cells had approximately 50% lower adduct levels than parental LNCaP and vector control cells. Bioactivation assays using LNCaP cytosols showed that enzymatic activation of N-OH-PhIP to a DNA binding species was dependent on ATP and could be inhibited by recombinant human GSTP1 in the presence of glutathione. This evidence confirms that N-OH-PhIP can be bioactivated to a DNA binding species in human prostate and human prostate-derived cells. These observations provide the basis for using LNCaP and LNCaP-GSTP1 cells as a model system for studying the role of this enzyme in protection against N-OH-PhIP induced DNA damage in prostate carcinogenesis. Loss of GSTP1 expression in human prostate may, therefore, enhance its susceptibility to carcinogenic insult by compounds such as N-OH-PhIP. Conversely, induction of GSTs in early-stage prostate carcinogenesis may be a useful protective strategy.

Human prostate cancer and benign prostatic hyperplasia: molecular dissection by gene expression profiling.

Critical aspects of the biology and molecular basis for prostate malignancy remain poorly understood. To reveal fundamental differences between benign and malignant growth of prostate cells, we performed gene expression profiling of primary human prostate cancer and benign prostatic hyperplasia (BPH) using cDNA microarrays consisting of 6500 human genes. Frozen prostate specimens were processed to facilitate extraction of RNA from regions of tissue enriched in either benign or malignant epithelial cell growth within a given specimen. Gene expression in each of the 16 prostate cancer and nine BPH specimens was compared with a common reference to generate normalized measures for each gene across all of the samples. Using an analysis of complete pairwise comparisons of expression profiles among all of the samples, we observed clearly discernable patterns of overall gene expression that differentiated prostate cancer from BPH. Further analysis of the data identified 210 genes with statistically significant differences in expression between prostate cancer and BPH. These genes include many not recognized previously as differentially expressed in prostate cancer and BPH, including hepsin, which codes for a transmembrane serine protease. This study reveals for the first time that significant and widespread differences in gene expression patterns exist between benign and malignant growth of the prostate gland. Gene expression analysis of prostate tissues should help to disclose the molecular mechanisms underlying prostate malignant growth and identify molecular markers for diagnostic, prognostic, and therapeutic use.

Cyclooxygenase-2 is up-regulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma.

Cyclooxygenase-2 (COX-2) is the inducible isoform of the rate-limiting enzymes that convert arachidonic acid to proinflammatory prostaglandins as well as a primary target for nonsteroidal anti-inflammatory drugs. Accumulating evidence suggests that up-regulation of COX-2 is associated with carcinogenesis in multiple organ systems including the large bowel, lung, breast, and prostate. In this report, we examine the expression of COX-2 protein and mRNA in prostate tissue containing various lesions and in prostate cancer cell lines. In the cell lines, LNCaP, DU145, PC-3, and TSU, COX-2 protein expression was undetectable under basal conditions but could be induced transiently by phorbol ester treatment in PC-3 and TSU cells, but not in DU145 and LNCaP cells. Immunohistochemical analysis of 144 human prostate cancer cases suggested that, in contrast to several previous reports, there was no consistent overexpression of COX-2 in established prostate cancer or high-grade prostatic intraepithelial neoplasia, as compared with adjacent normal prostate tissue. Positive staining was seen only in scattered cells (<1%) in both tumor and normal tissue regions but was much more consistently observed in areas of proliferative inflammatory atrophy, lesions that have been implicated in prostatic carcinogenesis. Staining was also seen at times in macrophages. Western blotting and quantitative RT-PCR analyses confirmed these patterns of expression. These results suggest that if nonsteroidal anti-inflammatory drugs are indeed chemopreventive and/or chemotherapeutic for prostate cancer, their effects are likely to be mediated by modulating COX-2 activity in non-PCa cells (either inflammatory cells or atrophic epithelial cells) or by affecting a COX-2-independent pathway.

Increasing incidence of minimal residual cancer in radical prostatectomy specimens.

Residual cancer in radical prostatectomy specimens from men with biopsy-proven adenocarcinoma occasionally may be difficult, or even impossible, to identify. Although this finding was recently described as "minimal residual cancer" or the "vanishing cancer phenomenon," there are no data on the incidence of this phenomenon in surgical pathology practice. We evaluated 3,038 consecutive radical prostatectomies performed at the Johns Hopkins Hospital between 1988 and 1995, excluding cases with a history of transurethral resection, prior therapy with a luteinizing hormone-releasing hormone agonist, focal Gleason grade 4 or 5, capsular penetration, or a positive surgical margin. Of this group, 84 cases with minimal or no residual cancer were identified. In 60 of these cases, residual cancer was "difficult to find" (mean total volume, 0.03 cc; range, 0.01-0.08 cc); in 20 cases, residual cancers were classified as "minute" (mean total volume, 0.07 cc; range, 0.03-0.13 cc). In four cases, no residual cancer could be identified, including two cases in which the diagnosis of cancer on needle biopsy was confirmed, one case in which review of the diagnostic needle biopsy revealed only high-grade prostatic intraepithelial neoplasia, and one case in which molecular analysis demonstrated mislabeling of the needle biopsy specimen. The annual incidence of minimal residual cancer increased from 0.5% in 1988 to 4% in 1993 and has begun to plateau at 3 to 4% since 1993 (p = 0.0016 for increasing trend). These data confirm the general impression that with more vigilant screening of men for prostate cancer, there has been an associated increase in cancer with little or no residual cancer at radical prostatectomy.

Histology and cellular kinetics of prostatic atrophy.

This study addresses two issues regarding prostatic atrophy: (1) the histologic features of atrophy as seen on needle biopsy results and how they affect the diagnosis of atrophy and (2) the cellular kinetics of atrophy and what it suggests about the mechanism of atrophy. We reviewed hematoxylin and eosin sections for 103 prostate needle biopsy specimens with atrophy. Each biopsy specimen was classified as either simple atrophy (large atrophic glands without crowding: 53 cases) or postatrophic hyperplasia (PAH) (crowded focus of small atrophic acini: 50 cases). Cell proliferation in both the atrophic and benign glands was evaluated in 103 cases by immunohistochemistry using antibodies against MIB-1. The TdT-mediated dUTP-biotin nick-end labeling technique was performed on 61 cases to quantitate apoptosis in atrophic and benign glands. Thirty-two percent of cases showed chronic inflammation, 21% showed acute inflammation, 14% showed nucleoli, and 1% showed mitoses. In comparison to simple atrophy, PAH contained more frequent prominent nucleoli (p < 0.0001) and acute inflammation (p < 0.0001), yet not chronic inflammation. In a multivariate analysis, acute inflammation and PAH pattern influenced the presence of prominent nucleoli. Staining for MIB-1 was greater in atrophic (27.5 cells/1000 cells) than in benign glands (3.5 cells/1000 cells), greater in PAH than in simple atrophy (p = 0.0015), and greater with acute (p = 0.05) but not chronic inflammation. In a multivariate analysis, only the pattern of atrophy and not acute inflammation was found to influence MIB-1. The rate of apoptosis was negligible in both the benign and atrophic glands, did not vary with pattern of atrophy, and did not correlate with MIB-1. Despite the atrophic appearance, atrophic glands in PAH show more proliferative activity than benign, nonatrophic glands and show no evidence of active involution, justifying the term "postatrophic hyperplasia" for this pattern of atrophy. Prominent nucleoli are seen more frequently in postatrophic hyperplasia, even in the absence of acute inflammation. To avoid a potential erroneous diagnosis of cancer, a constellation of features suggestive of malignancy should be considered, rather than relying on prominent nucleoli as the sole criteria for the diagnosis of prostate cancer.

Correlation of prostate needle biopsy and radical prostatectomy Gleason grade in academic and community settings.

Prior studies have not analyzed grading patterns and accuracy in nonacademic sites and have not analyzed reasons for discrepant grades. We analyzed 499 radical prostatectomy (RP) specimens at The Johns Hopkins Hospital (JHH) from 1994 and compared them with the corresponding JHH needle biopsy Gleason grade and, when available (n = 390), to the outside institution (non-JHH) Gleason biopsy grade. For JHH, there was exact agreement between biopsy and RP in 58% and agreement to within one digit in 93% of cases, compared with 34% and 67%, respectively, for non-JHH. Combining cases into more meaningful groups (Gleason 2-4, 5-6, 7, and 8-10), there was 66% exact correlation between the biopsy and RP for JHH as compared with 45% for non-JHH. Non-JHH undergraded biopsy results more than JHH, with 22.3% and 1.2% Gleason score 2-4, respectively. None of the cases with a Gleason score of 2-4 on biopsy from JHH or non-JHH were Gleason score 2-4 on RP. Fifty-five percent of the tumors with non-JHH needle biopsy results graded Gleason 2-4 had either capsular penetration, seminal vesicle, or lymph node involvement. All of the tumors with needle Gleason score 2-4 at JHH were organ confined. The JHH needle biopsy grade correlated better with pathologic stage than did non-JHH (R = 0.27 JHH vs. R = 0.12 non-JHH). Extent of cancer in the biopsy sample was not a factor in the accuracy of predicting RP grade or stage. Eighty-two cases evaluated at JHH were signed out by a genitourinary pathologist; the grading of the biopsy samples by other JHH pathologists was just as accurate. Gleason score of > or = 7 on the biopsy sample predicted a Gleason score of > or = 7 in the RP 87.5% of the time. A Gleason score of < 7 predicted a Gleason score of < 7 only 63.9% of the time. Discordant grades in some cases reflected patterns of cancer on needle biopsy that were borderline between two different Gleason scores. Sampling was the major source of discrepancy and was often due to the high-grade component in the RP not being present in the biopsy results or due to a component of cancer on the needle reflecting such a small percentage of the pattern seen on RP that this pattern was not included in the final RP Gleason score.

Prediction of progression following radical prostatectomy. A multivariate analysis of 721 men with long-term follow-up.

We studied 721 men with clinically confined disease who underwent radical prostatectomy. No patient received preoperative or postoperative radiotherapy or hormone therapy until progression occurred. For those men without progression, the mean follow-up was 6.5 years with a median of 6 years (range 1 to 12 years). Because patients with lymph node metastases or seminal vesicle invasion had such a high risk of progression, enhanced prognostication was not needed in men with these findings. Thus we focused this analysis on the 617 men without lymph node metastases or seminal vesicle invasion. In the multivariate analysis, Gleason score (P < 0.0001), surgical margins (P = 0.004), and capsular penetration (P = 0.007) were all independent predictors of progression. Tumors with a Gleason score of 2 through 4 were almost invariably cured, with a 10-year progression-free risk of 96%. At the opposite end of the spectrum, the 10-year actuarial progression-free risk for men with a Gleason score of 8 through 9 was 35%. Men with Gleason score 2 through 4 or 8 through 9 tumors could not be stratified into different risks of progression based on the presence or extent of capsular penetration or margin status. For the men with Gleason score 5 through 7 tumors (88.2% of cases), predicting their risk of progression was enhanced by knowledge of their tumor's capsular penetration and margin status. Tumors with a Gleason score of 5 through 6 and 7 were each stratified into three groups with different risks of progression. Using the actuarial curves within this study, physicians will be able to more accurately determine a patient's risk of progression following radical prostatectomy based on a combination of the radical prostatectomy Gleason score, extent of capsular penetration, and status of surgical margins of resection.

Ability of sextant biopsies to predict radical prostatectomy stage.

OBJECTIVES: There are few studies evaluating multiple variables on sextant biopsies with the intent to predict stage in radical prostatectomy specimens. METHODS: We studied 113 sextant biopsies with corresponding totally submitted radical prostatectomy specimens. Variables evaluated on sextant biopsies included total length and percent of cancer; maximum length and percent of cancer on one core; location (apex, mid, base); bilaterality; Gleason grade; number of cores involved; serum prostate-specific antigen (PSA) level; and serum PSA density (PSAD). Radical prostatectomy stage was classified as organ versus non-organ confined. RESULTS: The following variables individually correlated with radical prostatectomy stage: total cancer measured in millimeters (P <0.0001) or percent (P <0.0005); biopsy Gleason score (P <0.0001); number of involved cores (P <0.0001); maximum cancer on one core measured in millimeters (P = 0.0001); maximum percent of cancer on one core (P = 0.01); bilaterality (P = 0.01); PSA level (P = 0.03), and PSAD (P = 0.001). The most predictive sets of two variables that correlated with stage included high Gleason score (P <0.0001) combined with numbers of cores involved (P = 0.002). When biopsies had Gleason scores of 6 or less, two or fewer positive cores, and serum PSA of 0 to 4 ng/mL, 89% were organ confined. When biopsies had Gleason scores of 6 or less with two unilaterally positive cores, 87% were organ confined. In biopsies with Gleason scores of 7 or more and more than one positive core, only 10% were organ confined. CONCLUSIONS: The most important predictors of stage by sextant needle biopsy evaluation are numbers of cores involved with carcinoma and high Gleason score. Bilaterality and serum PSA values improved prediction in two small subgroups. In 37% of our population we were able to predict with a greater than 87% probability the organ-confined versus non-organ-confined status.

Histopathologic characterization of hereditary benign prostatic hyperplasia.

OBJECTIVES: Recent studies suggest the presence of a hereditary form of benign prostatic hyperplasia (H-BPH). This study was undertaken to characterize the histopathologic features of BPH in these men. METHODS: Because study subjects with H-BPH were young (mean age 59 years) and had a large prostate (mean prostate weight 61 g), we compared the histopathologic findings in these men with those in two different control groups: (1) age-matched control subjects (mean age 59 years; mean prostate weight 31 g), and (2) prostate weight-matched control subjects (mean age 70 years; mean prostate weight 61 g). Using a color video image analysis system, we morphometrically determined stromal/epithelial ratios in histologic sections taken from 12 men with H-BPH, 36 age-matched control subjects, and 36 prostate weight-matched control subjects. RESULTS: The stromal/epithelial ratio was 2.6 +/- 1.4 in the men with H-BPH, 2.7 +/- 1.7 in the age-matched control subjects, and 1.7 +/- 0.9 in the prostate weight-matched control subjects. Regression analysis, which controlled for the differences in prostate weight or patient age between men with H-BPH and age-matched and prostate weight-matched control subjects, respectively, revealed a significant difference between men with H-BPH and prostate weight-matched control subjects (P = 0.015) but no difference from age-matched control subjects (P = 0.36). CONCLUSIONS: The larger prostates in young men with H-BPH are characterized by a higher stromal/epithelial ratio than are similar-sized prostates in older men with sporadic BPH. This finding gives rise to speculation that H-BPH is associated with an increase in stromal elements.

Radical prostatectomy in men less than 50 years old.

We studied the effect of age on tumor progression (defined as postoperative prostate specific antigen elevation) in 543 men who underwent radical prostatectomy for clinically localized prostate cancer. Patients were divided into two age groups: patients under 50 years (N = 85) and patients older than 50 years (N = 458). The mean follow-up for both groups was S.3 years. Clinical stage was similar in both groups, with only 3% in each group detected by screening techniques. By Kaplan-Meier analysis, men under 50 years showed slightly less progression than men older than 50 years (p = 0.04), especially during the first 5 years following surgery. The key differentiating feature was a lower incidence of positive margins in the younger age group (18.8%) than the older age group (42.6%; p < 0.0001). There was a higher incidence of lymph node metastasis in the younger age group (14.1%) than the older age group (6.1%; p = 0.01); this adverse feature was present in only a small fraction of the patients and did not play a major role in the difference in progression between age groups. There was no statistically significant difference between the two age groups in tumor grade, capsular penetration, or seminal vesicle invasion. Gland volume was significantly higher in the older age group. Within the younger age group, progression was not affected by a family history of prostate cancer. We found that, despite the tendency in younger men to preserve the neurovascular bundles and cut closer to the prostate, this age group still has a lower incidence of positive surgical margins possibly due to greater ease of surgical removal of small glands. Young men who are candidates for radical prostatectomy do not have a worse prognosis following surgery than older men, and even fare better during the first five postoperative years.

Do close but negative margins in radical prostatectomy specimens increase the risk of postoperative progression?

PURPOSE: When tumor extends close to the margin of resection yet does not extend to the inked edge of the gland, it is unclear whether patients have an adverse prognosis compared to cases with greater distance between the tumor and margin. MATERIALS AND METHODS: Among radical prostatectomy specimens with negative margins the distance between the most peripheral tumor and the surgical margin of resection was measured in 52 cases with and 49 without progression. All patients had clinically confined disease (stages T1 or T2) with subsequent progression or a minimum 5-year followup without evidence of disease. No patient received preoperative or postoperative radiotherapy or hormonal therapy until progression occurred. All men underwent a postoperative serum prostate specific antigen test to evaluate progression. Seminal vesicles and lymph nodes were pathologically free of tumor. All prostates were serially sectioned, completely embedded and assessable regarding margins of resection. RESULTS: Patients with progression were no more likely to have tumor close to the margin than those without progression. In a regression analysis analyzing the effect of Gleason score, distance between tumor and margin, location of closest margin and pathological stage as related to progression, only grade was predictive of progression (p < 0.00001). CONCLUSIONS: It is not necessary for pathologists to designate these margins as close, since biologically this finding has no significance. Furthermore, physicians who are involved in treatment of patients after radical prostatectomy for prostate cancer should not alter therapy depending on whether margins are reported as close.

Immunoreactivity for prostate-specific antigen and prostatic acid phosphatase in adenocarcinoma of the prostate: relation to progression following radical prostatectomy.

BACKGROUND: Although, in general, immunoperoxidase staining for prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) cannot distinguish between benign and malignant prostatic epithelium, immunoreactivity of these antigens may be helpful in predicting prognosis of prostate cancer. The purpose of this study was to evaluate intensity and extent of immunoperoxidase staining for PSA and PSAP as a prognostic tool in prostate adenocarcinomas. METHODS: We studied radical prostatectomy specimens from 68 patients with the following stages: organ-confined, 34.3%; focal capsular penetration, 38.8%; established capsular penetration, 25.3%; and seminal vesicle invasion, 1.6%. Ninety-one percent of cases were Gleason score 5-7. The mean follow-up for those men without progression was 8.9 years, compared to 3.5 years for those with progression. Progression was defined as an elevated postoperative serum PSA level (> 0.2 ng/ml). Intensity of PSA and PSAP staining was recorded and based on a scale of 0-3 (0, no staining; 1, weak; 2, moderate; 3, intense). Extent was quantitated on a scale of 0-4 (0, 0-5% staining; 1, 6-35%; 2, 36-65%; 3, 65-95%; 4, 95-100%). A score (0-12) was computed by multiplying intensity and extent of the stain in the tumor area. RESULTS AND CONCLUSIONS: Intensity and extent of PSA and PSAP immunoreactivity did not predict progression in adenocarcinomas of the prostate following radical prostatectomy.

Cell proliferation and apoptosis in prostate cancer--correlation with pathologic stage?

Gleason grade, especially at low and high ends of the spectrum, is a known predictor of pathologic stage. What are needed are predictors of stage with Gleason sum 5 to 7 tumors, which encompasses the majority of clinically organ-confined tumors. In this study, we analyzed whether cell proliferation and apoptosis (programmed cell death) were correlated with stage in men with clinically organ-confined Gleason sum 6 to 7 cancer. We studied 98 radical prostatectomies with the following pathologic stages: organ-confined disease (n = 28); capsular penetration (n = 28); seminal vesicle invasion (n = 21); and pelvic lymph node metastases (n = 21). Histological sections from the radical prostatectomies were stained for cell proliferation using MIBI antibody (Ki-67) and for apoptosis using the TUNEL technique. The extent of staining was recorded as the number of positive cells per 1000 cells. Overall daily growth (kp) was calculated as: kp = (ki/2) - (apoptosis/0.5), based on the time of prostate cancer to undergo mitosis and apoptosis per day. Using logistic regression analysis, pathologic stage did not correlate with cell proliferation, apoptosis, or overall daily growth. These parameters also did not distinguish between Gleason sum 6 and Gleason sum 7 tumors. We supplemented these cases with examples of Gleason sum < or = 4 and Gleason sum > or = 8 to study cell proliferation and cell death in the full spectrum of Gleason grades. There was a significant difference (P = 0.005) in cell proliferation between Gleason sum > or = 6 and Gleason sum < or = 4 tumors, but apoptosis and daily growth were not significant. We conclude that cell proliferation and apoptosis do not correlate with pathological stage in clinically organ-confined cancer with Gleason sum 6 or 7, but that cell proliferation can distinguish between high (Gleason sum > or = 6) and low (Gleason sum < or = 4) grade tumors.

Use of repeat sextant and transition zone biopsies for assessing extent of prostate cancer.

PURPOSE: Little is known why certain prostate cancers are missed on biopsy. In patients with a needle biopsy diagnosis of cancer it is also unknown whether repeat needle biopsy provides useful information to predict extent of disease. MATERIALS AND METHODS: In the pathology laboratory we performed sextant and transition zone needle biopsies on 193 radical prostatectomy specimens from men with nonpalpable cancer detected on needle biopsy (stage T1c) using an 18 gauge biopsy gun. Radical prostatectomy specimens were then serially sectioned, totally embedded, mapped and staged. RESULTS: The transition zone biopsy by itself was positive in only 2.1% of cases, demonstrating the lack of usefulness for this particular biopsy. Despite cancer on preoperative needle biopsy in all cases, 31% showed no cancer on repeat sextant transition zone biopsy. In a multivariate analysis (variables included radical prostatectomy tumor volume, radical prostatectomy tumor location, prostate gland size and radical prostatectomy grade) decreased tumor volume (p < 0.0001), increased gland size (p = 0.001), and decreased radical prostatectomy grade (p = 0.013) were each independent predictors of absence of tumor on repeat biopsy. A lack of cancer on repeat biopsy correlated with pathological stage: 90% of cases without cancer on repeat biopsy were organ confined versus 66% for cases with a single less than 3 mm. focus of cancer on repeat biopsy versus 58% for cases with more cancer on repeat biopsy. Of 38 men with a preoperative needle biopsy showing less than 3 mm. of cancer on 1 core that was not high grade and with prostate specific antigen 10 or less (men for whom urologists are most likely to repeat biopsy) the presence of cancer on repeat biopsy also correlated with extent of disease at radical prostatectomy. However, of these 38 men 6 of 16 with no cancer on repeat biopsy had moderate tumor (4 with organ confined Gleason score 5 to 6, tumor volume 0.79 to 4.5 cc; 1 with organ confined Gleason score 7, tumor volume 0.18 cc; and with 1 established penetration Gleason score 6, tumor volume 0.53) at radical prostatectomy. CONCLUSIONS: Although absence of cancer on repeat biopsy correlates with various parameters of extent of disease, there is significant overlap for the individual patient. This study also demonstrates the limits of sextant needle biopsy to evaluate tumor status in patients who elect watchful waiting or less invasive forms of therapy (cryotherapy, interstitial radiotherapy).

Prospective evaluation of men with stage T1C adenocarcinoma of the prostate.

PURPOSE: The pathological characteristics of stage T1c cancers in the era of widespread prostate specific antigen (PSA) testing were determined, and the ability of pretreatment parameters to predict tumor significance in men with stage T1c disease was evaluated. MATERIALS AND METHODS: Of 336 men with stage T1c prostate cancer seen between 1994 and 1996, 240 (71.4%) were treated with radical prostatectomy, 20 (6%) with radiation therapy and 76 (22.6%) expectantly. Recommendations for treatment were based on previously determined criteria predictive of a significant stage T1c cancer (more than 0.2 cm.3): 1) PSA density 0.15 ng./ml./gm. or more, 2) Gleason score 7 or greater, 3) 3 or more cores involved with cancer, or 4) 50% or more involvement of any core with cancer. Pathological evaluation of prostatectomy specimens allowed classification of tumors as insignificant (confined tumor smaller than 0.2 cm.3 with a Gleason score of less than 7), minimal (confined tumor 0.2 to less than 0.5 cm.3 with a Gleason score of less than 7), moderate (0.5 cm.3 or larger disease, or capsular penetration with a Gleason score of less than 7) and advanced (capsular penetration with a Gleason score of 7 or more, or positive margins, seminal vesicles or lymph nodes). Pathological characteristics of tumors in this series were compared to a previous series of 157 men with stage T1c cancers who underwent radical prostatectomy between 1988 and 1992. RESULTS: Of 240 men who underwent radical prostatectomy tumors were insignificant in 40 (17%), minimal in 29 (12%), moderate in 124 (52%) and advanced in 47 (19%). An increase in organ confined cancers (51 to 72%) and a decrease in positive margins (17 to 8%) were noted when comparing stage T1c series (1988 to 1992 versus 1994 to 1996) but the percentage of insignificant tumors remained stable (16 versus 17%) between series. Ultrasound and sextant biopsies were available for review in 72 cases (current series). If the pretreatment criteria used to recommend therapy suggested significant tumor (64 cases) then insignificant tumor was present in only 10 (16%). If pretreatment criteria suggested insignificant tumor (8 cases), insignificant or minimal tumor was present in 6 (75%) and moderate organ confined disease was present in 2 (25%). The absence of a lesion on ultrasound and measurement of total length of cancer within the biopsy specimen were not predictive of an insignificant tumor. CONCLUSIONS: In a nonscreened population stage T1c cancers are being discovered earlier with widespread PSA testing. Even with the detection of earlier cancers we demonstrated that it is possible to minimize the number of patients with small tumors who will undergo radical prostatectomy using pretreatment criteria to counsel men regarding appropriate management options.

Detection and analysis of beta-catenin mutations in prostate cancer.

BACKGROUND: E-cadherin and alpha-catenin are components of adherens junctions which mediate calcium-dependent, cell-cell adhesion in a homotypic manner. Both these molecules have been defined as useful tumor markers as their altered expression correlates with increased tumor aggressiveness and dedifferentiation. More recently, alterations of a third component of adherens junctions, beta-catenin, have been observed to play a role in several human cancers. Dysregulation of beta-catenin, either by direct mutation or by defects in interacting pathways/regulators, can result in its cytoplasmic accumulation and nuclear translocation. In the nucleus, beta-catenin forms a transcriptional complex capable of upregulating target genes, many of which encode proliferative factors. Given its oncogenic activity and connection to human cancer, we examined the beta-catenin gene and its expression in prostate cancer. METHODS: By single-stranded conformational polymorphism (SSCP) and DNA sequencing analyses, we screened exon 3 of beta-catenin from a panel of 81 primary tumors obtained at radical prostatectomy, 22 lymph node metastases from untreated patients, and a unique set of 61 metastatic tissues from 19 patients who died of hormone-refractory disease. RESULTS: We found putative activating mutations (missense and deletion) at a rate of 5% (7/138). One patient had the same 72 base pair deletion in each of nine separate metastases examined, indicating that this change was associated with a clonal population of metastatic cells. CONCLUSIONS: Immunohistological staining of mutation-positive tumors demonstrated beta-catenin accumulation and nuclear localization in a heterogeneous fashion. Consistent with this in vivo finding, our in vitro analyses demonstrate that certain mutations can result in increased beta-catenin nuclear activity in prostate cancer cell lines. These data implicate the beta-catenin signaling pathway in the development of a subset of prostate cancers.

CD44 and CD44v6 downregulation in clinical prostatic carcinoma: relation to Gleason grade and cytoarchitecture.

BACKGROUND: Altered expression of CD44 has been implicated in tumor progression and metastasis in multiple neoplasms. METHODS: CD44 expression in archival tissues of prostate carcinoma was examined by immunohistochemistry with monoclonal antibodies against core CD44 and the RNA splice variant CD44v6 (v6). RESULTS: Core CD44 expression was reduced in the majority of primary neoplastic foci (n = 94) and loss of expression correlated with increasing Gleason grade. Staining for v6 was absent in most carcinomas and metastases. Expression of core CD44 in pelvic lymph node (n = 27) and bone metastases (n = 21) was significantly reduced. In addition, CD44 expression correlated with cytoarchitecture. Tall columnar tumor cells typically stained positively, yet more rounded cells forming cribiform structures or nests showed reduced expression. All cases of high-grade prostatic intraepithelial neoplasia were positive for core CD44 yet, there was decreased expression in cribiform and micropapillary variants. CONCLUSIONS: The majority of clinically relevant human prostatic carcinomas and metastases downregulate expression of CD44. Additional studies to determine whether CD44 cell surface expression relates to clinical outcome independent of other established clinicopathologic risk factors are warranted.

Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in prostate cancers.

Proteins encoded by bcl-2 family genes are important regulators of programmed cell death and apoptosis. Alterations in the expression of these apoptosis-regulating genes can contribute to the origins of cancer, as well as adversely influence tumor responses to chemo- and radiotherapy. Using antibodies specific for the Bcl-2, Bax, Bcl-X, and Mcl-1 proteins in combination with immunohistochemical methods, we examined for the first time the expression of these bcl-2 family genes in 64 cases of adenocarcinoma of the prostate, including 10 Gleason grade 2 to 4 tumors, 21 grade 5 to 7 tumors, 17 grade 8 to 10 tumors, 8 lymph node metastases, and 8 bone metastases. In addition, 24 cases of prostatic intraepithelial neoplasia (PIN) or PIN coexisting with carcinoma were also evaluated. All immunostaining results were scored with regard to approximate percentage of positive tumor cells and relative immunostaining intensity. Expression of the anti-apoptotic protein Bcl-2 was present in 16 of 64 (25%) adenocarcinomas and tended to be more frequent in high grade tumors (Gleason grade 8 to 10; 41%) and nodal metastases (38%) than in lower grade (Gleason 2 to 7) primary tumors (16%; P < 0.05). Bcl-X was expressed in all 64 (100%) tumors evaluated. Bcl-X immunointensity was generally stronger in high grade primary tumors (grade 8 to 10) and metastases compared with PIN and low grade neoplasms (P < 0.0001). In addition, the proportion of specimens with > 50% Bcl-X-immunopositive tumor cells also was higher in advanced grade primary tumors (Gleason 8 to 10) and metastases than in PIN and low grade tumors (Gleason 2 to 7; P < 0.005). The anti-apoptotic protein Mcl-1 was expressed in 52 of 64 (81%) tumors, compared with only 9 of 24 (38%) cases of PIN (P < 0.001). In addition, the percentage of Mcl-1-positive cells was typically higher in Gleason grade 8 to 10 tumors and metastases than in PIN or lower grade tumors (P = 0.025). In contrast, the pro-apoptotic protein Bax was expressed in all prostate cancers evaluated, with high percentages of immunopositive cells and strong immunointensity typically occurring regardless of tumor grade. The findings suggest that expression of several anti-apoptotic members of the bcl-2 gene family, including bcl-2, bcl-X, and mcl-1 increases during progression of prostate cancers, a finding that may be relevant to the hormone-insensitive, metastatic phenotype of most advanced adenocarcinomas of the prostate.

Cell turnover in human seminal vesicles and the prostate: an immunohistochemical study.

The human prostate and seminal vesicles are both androgen-dependent sex accessory organs. Their growth behavior, response to hormone manipulation, susceptibility to benign and malignant processes and sex accessory functions, however, differ greatly. The growth behavior of most tissues correlates well with the cell turnover rate of that tissue. Therefore, we compared the cell turnover of normal human prostate and seminal vesicles. Immunohistochemical expression of MIB-1 (proliferation), bcl-2 and transforming growth factor (TGF beta) were examined in 20 different samples taken from histologically normal human prostatic and seminal vesicle tissue. For the quantification of apoptosis, the TUNEL technique was used. The apoptosis rates in normal prostatic tissue (0.73+/-0.60) were significantly greater (P=0.003) than those seen in seminal vesicles (0.02+/-0.01). The proliferation rates also differed significantly (P=0.002) between these tissues (prostate: 0.77+/-0.78; seminal vesicles: 0.02+/-0.02). Eighty percent of the prostate tissue stained for bcl-2, whereas only 55% of the seminal vesicle tissue showed staining for bcl-2. All seminal vesicles and 75% of the prostate samples stained for TGF beta. For both androgen-dependent tissues, apoptotic rates closely equaled proliferation rates. The cell turnover, however, was much higher in the prostate than in the seminal vesicles. TGF beta seems to be more important for the regulation of cell turnover in the seminal vesicles than bcl-2. These differences in the proliferative behavior may explain why disturbances of apoptotic regulation lead to a more extensive net cell gain in prostatic tissue compared to the seminal vesicles. This might help explain the vastly different incidence of benign and malignant tumors in these organs.