Association of HLA polymorphisms with post-transplant lymphoproliferative disorder in solid-organ transplant recipients.

The association between HLA polymorphisms and PTLD was investigated in a case-control study, comparing 110 predominantly adult solid-organ transplant recipients who developed PTLD to 5601 who did not. Donor and recipient HLA were analyzed. We detected a significant association between recipient HLA-A26 and the development of PTLD (OR 2.74; p = 0.0007). In Caucasian recipients, both recipient and donor HLA-A26 were independently associated with development of PTLD (recipient A26 OR 2.99; p = 0.0004, donor A26 OR 2.81; p = 0.002). Analysis of HLA-A and -B haplotypes revealed that recipient HLA-A26, B38 haplotype was strongly correlated with a higher incidence of EBV-positive PTLD (OR 3.99; p = 0.001). The common ancestral haplotype HLA-A1, B8, DR3, when carried by the donor, was protective against PTLD (OR 0.41; p = 0.05). Several other HLA specificities demonstrated associations with clinical and pathological characteristics as well as survival. These findings demonstrate the importance of HLA polymorphisms in modulating the risk for PTLD, and may be useful in risk stratification and development of monitoring and prophylaxis strategies.

Annexin-V imaging for noninvasive detection of cardiac allograft rejection.

Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.

T cells reactive to an inducible heat shock protein induce disease in toxin-induced interstitial nephritis.

T cells reactive against immunodominant regions of inducible heat shock proteins (HSPs) have been identified in the chronic inflammatory lesions of several experimental autoimmune diseases. Since HSPs are known to be induced by a number of renal tubular epithelial cell toxins associated with chronic interstitial nephritis, we investigated the relevance of HSP expression and T cell reactivity to HSP70 in a model of progressive inflammatory interstitial nephritis. Chronic administration of cadmium chloride (CdCl2) to SJL/J mice induces HSP70 expression in renal tubular cells 4-5 wk before the development of interstitial mononuclear cell infiltrates. CdCl2 also induces HSP70 expression in cultured tubular epithelial cells from SJL/J mice. CD4+, TCR-alpha/beta+ T cell lines specific for an immunodominant HSP peptide are cytotoxic to heat stressed or CdCl2-treated renal tubular cells. Such HSP-reactive T cells mediate an inflammatory interstitial nephritis after adoptive transfer to CdCl2-treated mice at a time when immunoreactive HSP70 is detectable in the kidneys, but before the development of interstitial mononuclear cell infiltrates. T cells isolated from the nephritic kidneys of mice treated with CdCl2 for 13 wk are also cytotoxic to heat shocked or cadmium-treated tubular cells. These kidney-derived T cells additionally induced interstitial nephritis after passive transfer, indicating their pathogenic significance. Our studies strongly support a role for HSP-reactive T cells in CdCl2-induced interstitial nephritis and suggest that the induction of HSPs in the kidney by a multitude of "non-immune" events may initiate or facilitate inflammatory damage by HSP-reactive lymphocytes.

Identification and characterization of a fibroblast marker: FSP1.

We performed subtractive and differential hybridization for transcript comparison between murine fibroblasts and isogenic epithelium, and observed only a few novel intracellular genes which were relatively specific for fibroblasts. One such gene encodes a filament-associated, calcium-binding protein, fibroblast-specific protein 1 (FSP1). The promoter/enhancer region driving this gene is active in fibroblasts but not in epithelium, mesangial cells or embryonic endoderm. During development, FSP1 is first detected by in situ hybridization after day 8.5 as a postgastrulation event, and is associated with cells of mesenchymal origin or of fibroblastic phenotype. Polyclonal antiserum raised to recombinant FSP1 protein stained the cytoplasm of fibroblasts, but not epithelium. Only occasional cells stain with specific anti-FSP1 antibodies in normal parenchymal tissue. However, in kidneys fibrosing from persistent inflammation, many fibroblasts could be identified in interstitial sites of collagen deposition and also in tubular epithelium adjacent to the inflammatory process. This pattern of anti-FSP1 staining during tissue fibrosis suggests, as a hypothesis, that fibroblasts in some cases arise, as needed, from the local conversion of epithelium. Consistent with this notion that FSP1 may be involved in the transition from epithelium to fibroblasts are experiments in which the in vitro overexpression of FSP1 cDNA in tubular epithelium is accompanied by conversion to a mesenchymal phenotype, as characterized by a more stellate and elongated fibroblast-like appearance, a reduction in cytokeratin, and new expression of vimentin. Similarly, tubular epithelium submerged in type I collagen gels exhibited the conversion to a fibroblast phenotype which includes de novo expression of FSP1 and vimentin. Use of the FSP1 marker, therefore, should further facilitate both the in vivo studies of fibrogenesis and the mapping of cell fate among fibroblasts.

Limitations of metabolic activation systems used with in vitro tests for carcinogens.

Important differences between the metabolic activation of 7,12-dimethylbenz[a]anthracene in intact cellular systems and in liver homogenates suggest that the use of homogenates in conjunction with short-term assays for carcinogens could yield misleading results.

Induction of donor-specific unresponsiveness by intrathymic islet transplantation.

The application of isolated pancreatic islet transplantation for treatment of diabetes mellitus has been hampered by the vulnerability of islet allografts to immunologic rejection. Rat islet allografts that were transplanted into the thymus of recipients treated with a single injection of anti-lymphocyte serum survived indefinitely. A state of donor-specific unresponsiveness was achieved that permitted survival of a second donor strain islet allograft transplanted to an extrathymic site. Maturation of T cell precursors in a thymic microenvironment that is harboring foreign alloantigen may induce the selective unresponsiveness. This model provides an approach for pancreatic islet transplantation and a potential strategy for specific modification of the peripheral immune repertoire.

Resolution of nephrotic syndrome after successful bariatric surgery in patient with biopsy-proven FSGS.

The incidence of obesity-related nephropathy (ORG) is increasing with the growing incidence of obesity. ORG is associated with morbid obesity, proteinuria and renal biopsy findings of focal global and segmental glomerulosclerosis (FSGS), which can be associated with significant renal impairment. Weight reduction is associated with improvement of ORG, however, conservative measures aiming at long-term weight reduction are difficult to achieve. Bariatric surgery is the most effective way of achieving long-term weight reduction. We present a case of ORG with nephrotic-range proteinuria and FSGS on renal biopsy. Following bariatric surgery, patient achieved successful weight reduction with significant decrease in proteinuria and stabilization of renal function.

Nek3 kinase regulates prolactin-mediated cytoskeletal reorganization and motility of breast cancer cells.

Prolactin (PRL) stimulates the cytoskeletal re-organization and motility of breast cancer cells. During PRL receptor signaling, Vav2 becomes phosphorylated and activated, an event regulated by the serine/threonine kinase Nek3. Given the regulatory role of Vav2, the function of Nek3 in PRL-mediated motility and invasion was examined. Overexpression of Nek3 in Chinese hamster ovary transfectants potentiated cytoskeletal re-organization in response to PRL. In contrast, downregulation of Nek3 expression by small-interfering RNA (siRNA) attenuated PRL-mediated cytoskeletal reorganization, activation of GTPase Rac1, cell migration and invasion of T47D cells. In addition, PRL stimulation induced an interaction between Nek3 and paxillin and significantly increased paxillin serine phosphorylation, whereas Nek3 siRNA-transfected cells showed a marked reduction in paxillin phosphorylation. Analysis of breast tissue microarrays also demonstrated a significant up-regulation of Nek3 expression in malignant versus normal specimens. These data suggest that Nek3 contributes to PRL-mediated breast cancer motility through mechanisms involving Rac1 activation and paxillin phosphorylation.

Stabilization of prolactin receptor in breast cancer cells.

The role of the hormone prolactin (PRL) in the pathogenesis of breast cancer is mediated by its cognate receptor (PRLr). Ubiquitin-dependent degradation of the PRLr that negatively regulates PRL signaling is triggered by PRL-mediated phosphorylation of PRLr on Ser349 followed by the recruitment of the beta-transducin repeats-containing protein (beta-TrCP) ubiquitin-protein isopeptide ligase. We report here for the first time that interaction between PRLr and beta-TrCP is less efficient in human breast cancer cells than in non-tumorigenic human mammary epithelial cells. Furthermore, we demonstrate that both PRLr degradation and PRLr phosphorylation on Ser349 are impaired in breast tumor cells and tissues, an observation that directly correlates with enhanced expression of the PRLr in malignant breast epithelium. These findings represent a novel mechanism through which altered PRLr stability may directly influence the pathogenesis of breast cancer.

Sustained inhibition of intimal thickening. In vitro and in vivo effects of polymeric beta-cyclodextrin sulfate.

Intimal thickening after vascular injury may be modulated in part by heparin binding growth factors. We hypothesized that placement of a therapeutic polymer in the periadventitial space capable of tightly binding growth factors might alter the vascular response to injury. We first demonstrated that incubation of rat aortic smooth muscle cells with an insoluble, sulfated polymer of beta-cyclodextrin (P-CDS) was associated with a dose-dependent inhibition of proliferation induced by fetal calf serum, fibroblast growth factor-2 (FGF-2), platelet-derived growth factor BB, or epidermal growth factor. Preincubation studies of P-CDS with FGF-2 revealed a very rapid removal of mitogenic activity. Using radiolabeled FGF-2 (0.25 microg/ml), we observed a very rapid association rate (0.34 +/- 0.07 min-1, n=4) and a very slow dissociation rate (3.3 +/- 0.2 X 10(-7) min-1) at 37 degrees C, suggesting a high affinity interaction. Using both Transwell and linear under-agarose assays, we demonstrated a significant inhibition of random migration (chemokinesis) by P-CDS. Unsulfated polymeric beta-cyclodextrin (P-CD) had little if any of these effects, suggesting that the high negative charge density of P-CDS was important for the effects. Finally, rats undergoing carotid artery balloon injury were randomized to treatment with periadventitial P-CDS or no treatment, and were killed at 4 (n=20), 14 (n=59), and 88 d (n=14). Morphometric analysis demonstrated significant and sustained inhibition of intimal thickening in P-CDS-treated rats at 14 (P < 0.01) and 88 d (P < 0.05) using absolute intimal area or intima/media area ratios. No inhibition was seen in a group of rats treated with P-CD. In P-CDS-treated rats, bromodeoxyuridine labeling studies revealed fewer labeled smooth muscle cells in the intima at 14 d (P=0.01), while staining with Evans blue revealed enhanced late endothelial cell regrowth. Thus, periadventitially applied sulfated beta-cyclodextrin polymer, which can tightly bind heparin binding growth factors, inhibits intimal thickening in vivo in a sustained fashion without using an additional delivery system. These studies suggest that cellular processes mediated by heparin binding growth factors may be modulated by P-CDS.

Binding of radioactivity after transplacental administration of tritiated N-nitrosodiethylamine to Syrian golden hamsters.

Tritiated N-nitrosodiethylamine (DEN) was administered to two groups of female Syrian golden hamsters on days 11 and 15 of pregnancy. Binding of radioactivity was measured in maternal and fetal organs after various intervals by liquid scintillation counting of dehydrated and combusted tissues. No bound activity was found in the fetal tracheas on day 11 of gestation, when transplacental administration of DEN is known to be noncarcinogenic for the offspring. On day 15, when DEN administration caused a 95% incidence of tracheal tumors in the offspring, bound radioactivity was found in the fetal tracheas. In the respiratory tracts of the mothers, the distribution of bound radioactivity correlated with the distribution of target cell types. Binding was high in segmental bronchi and bronchioles which contain numerous Clara cells, the major source of DEN-induced pulmonary tumors. No binding occurred in the main bronchi, which do not possess Clara cells.

Biochemical basis for cytotoxicity of 7,12-dimethylbenz(a)anthracene in rat liver epithelial cells.

When the effects of 7,12-dimethylbenz(a)anthracene (DMBA) on normal and malignant rat liver epithelial cells were compared in a colony inhibition assay, this carcinogen showed a preferential cytotoxic action on the normal cells. In investigations of the biochemical basis of this selective toxicity, it was found that both cell lines were similarly effective in binding DMBA to DNA and that both cell lines had the capacity to metabolize this carcinogen. However, the hepatoma cells were more efficient than were the normal cells in generating very polar metabolites (not organic solvent extractable). These studies suggest that the basis of the resistance of the hepatoma cells to the toxicity induced by DMBA lies in their ability to detoxify biologically active metabolites. Several phenols were examined as possible toxic metabolites of DMBA, but these were not toxic at dose levels at which DMBA kills most of the normal cells.

Metabolism of 7,12-dimethylbmethylbenz(a)anthracene by macrophages and uptake of macrophage-derived metabolites by respiratory tissues in vitro.

Cultured mouse macrophages and tracheal and lung tissue each produced the same ethyl acetate-soluble derivatives of 7,12-dimethylbenz(a)anthracene (DMBA). The derivatives produced in the different cultures were indistinguishable by thin-layer chromatography and by high-pressure liquid chromatography but differed in their relative proportions. The greatest difference was seen between lungs and macrophages. The predominant metabolite produced by lungs was 8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz(a)anthracene, while macrophages produced equal quantities of both 8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz(a)anthracene and a second uncharacterized derivative, Metabolite B, at low DMBA doses (less than 0.05 microgram/ml medium) and primarily Metabolite B at higher DMBA doses (greater than 0.05 microgram/ml medium). Macrophages released the majority of the ethyl acetate-soluble metabolites that they produced into the surrounding medium. With the exception of 8,9-dihydro-8,9-dihydroxy-7,12dimethylbenz(a)anthracene, these derivatives were accumulated within tracheal and lung tissue when these organs were cocultivated with macrophages in the presence of DMBA.

Evaluation of metabolic activation of 7,12-dimethylbenz(a)anthracene in vitro by aroclor 1254-induced rat liver S-9 fraction.

Short-term assays for detection of chemical carcinogens frequently rely on an Aroclor 1254-induced rat liver S-9 fraction for metabolic activation of test compounds. The ability of this in vitro system to reproduce the activation occurring in target tissue was investigated by examining the DNA adducts produced when the polycyclic aromatic hydrocarbon carcinogen, 7,12-dimethylbenz(a)anthracene (DMBA), was incubated with the S-9 fraction and calf thymus DNA. Analyses by Sephadex LH-20 column chromatography of hydrocarbon-deoxyribonucleoside adducts obtained after enzymic digestion of the [3H]DMBA-modified DNA revealed that the products of binding of DMBA to DNA in the presence of the S-9 fraction vary with the relative concentration of DMBA to S-9 fraction. Further analyses of these adducts by high-pressure liquid chromatography in the presence of the diol-epoxide-DNA adduct (isolated from mouse embryo cells exposed to [14C]DMBA) and chemically synthesized ultraviolet-absorbing markers of DMBA 5,6-oxide-deoxyribonucleoside adducts showed that, at high DMBA-S-9 ratios, DMBA 5,6-oxide-deoxyriboiucleoside adducts were prominent among the products while, at low DMBA-S-9 ratios, the products included the diol-epoxide-DNA adduct found in target tissue. However, this adduct was always accompanied by other adducts not found in intact cellular systems. Inclusion of a metabolic inhibitor (1,1,1-trichloropropylene oxide) in the Salmonella mutagenicity assay demonstrated that high levels of revertants can be obtained from rat liver S-9 fraction-activated DMBA under conditions which should prohibit formation of the diol-epoxide. These results suggest that Aroclor 1254-induced rat liver S-9 fraction does not exactly reproduce the metabolic activation of this particular carcinogen in vivo and therefore should not be assumed to do this for other carcinogens.

Comparison of metabolism-mediated binding to DNA of 7-hydroxymethyl-12-methylbenz(a)anthracene and 7, 12-dimethylbenz(a)anthracene.

Comparison of the binding to DNA of 7-hydroxymethyl-12-methylbenza(a)anthracene and 7, 12-dimethylbenz(a)anthracene (DMBA) catalyzed by mouse embryo cells in culture or by rat liver microsomes indicates that the products formed are different for the two hydrocarbons. Thus, the hydroxy compound is not an intermediate in the binding of DMBA to DNA in these systems. Binding of the hydroxy compound to DNA in mouse embryo cells is less efficient than for DMBA and is inhibited by 1,1,1-trichloropropylene 2,3-oxide, an inhibitor of epoxide hydrase. This and the fluorescence spectra of the hydroxy compound-DNA adducts indicate that the hydroxy compound is activated for DNA binding through the formation of a diol-epoxide in the 1,2,3,4-ring. As previously found for DMBA, this is consistent with the activation of this compound through a bay-region diol-epoxide.

Cellular pharmacology of cyclopentenyl cytosine in Molt-4 lymphoblasts.

The toxicity, uptake, and metabolism of the oncolytic nucleoside cyclopentenyl cytosine (CPEC) have been examined in the Molt-4 line of human lymphoblasts. This compound is known to be converted to its 5'-triphosphate, which inhibits CTP synthetase and depletes the pools of cytidine nucleotides. In the Molt-4 system, the concentration of drug reducing proliferation by 50% in a 24-h incubation was between 50 and 100 nM. Cytidine, uridine, and nitrobenzylthioinosine almost fully prevented the cytotoxicity of CPEC when introduced shortly before or together with the drug, but only cytidine was effective as an antidote when added 12 h after 200 nM CPEC. Studies of the cellular entry of CPEC revealed that nitrobenzylthioinosine fully blocked this process over a 60-s interval and for as long as 2 h, suggesting that the initial interiorization was mediated by facilitated diffusion. In Molt-4 cells incubated with tritiated CPEC, 9 metabolites could be distinguished: prominent among these was cyclopentenyl uridine (CPEU), the deamination product of CPEC; other major metabolites included the 5'-mono-, di-, and triphosphates of CPEC, and of CPEU, along with two phosphodiesters provisionally identified as CPEC-diphosphate choline and CPEC-diphosphate ethanolamine. When the accumulation of CPEC-5'-triphosphate was measured as a function of concentration of the drug in the medium, the process was found not to be saturable by levels of CPEC up to 1000 nM. In cells incubated with 200 nM drug, CPEC-5'-triphosphate accumulated rapidly and linearly for approximately 4 h, the time for doubling of the concentration being 2 h. After a 16-h incubation with 100 nM CPEC, the concentration of CPEC-5'-triphosphate was 50-fold that of the parent drug in the medium and could be readily monitored spectrophotometrically in high-pressure liquid chromatography effluents without recourse to radiolabeled nucleoside. In 2-h incubations, the concentration of free CPEC required to reduce CTP by 50% was 150 nM; this corresponded to a CPEC-5'-triphosphate level of 750 nM. After washout of extracellular CPEC, CPEC-5'-triphosphate decayed with a half-life that ranged from 9 to 14 h. Twenty-four h after washout of 200 nM CPEC (the concentration of drug reducing proliferation by 80%), cells had not resumed proliferation, and CTP pools were still depressed by 90%. Cytidine, uridine, and nitrobenzylthioinosine all strongly repressed the anabolic phosphorylation of CPEC when added to Molt-4 cells along with the drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterization of the TERE1 gene, a gene down-regulated in transitional cell carcinoma of the bladder.

We have identified a novel cDNA product designated transitional epithelial response gene (TERE1), which was localized to chromosome 1p36. The TERE1 transcript (1.5 and 3.5 kb) is present in most normal human tissues including urothelium, but was reduced or absent in the majority of muscle invasive TCC tumors (22 out of 29 cases). The open reading frame encodes a protein of 338 amino acids (MW 36.8 KD). This protein is 57% homologous to a Drosophila protein called heix. We have shown by Western blotting and immuno-histochemistry with a polyclonal antibody to a specific TERE1 peptide, reduced or absent staining in muscle invasive tumors. Transfection of a sense TERE1 construct resulted in an 80-90% inhibition of cellular proliferation in two TCC cell lines and a lack of aneuploidy in the TERE1-transduced J82 cell line. These data suggest a potential role for this gene product in the progression of bladder cancer.

Novel p21WAF1/CIP1 mutations in superficial and invasive transitional cell carcinomas.

Alterations in the p53 gene are a predominant component in the development of transitional cell carcinoma (TCC), but the particular pathways distal to p53 alterations which contribute to urothelial transformation are not defined. Here, the p21WAF1/CIP1 gene, a p53 inducible and p53 independent gene product, was studied in TCC. p21WAF1/CIP1 expression was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) from five cell lines and 28 tumor specimens (14 superficial, 14 muscle invasive). This was expressed as a ratio of the gene product to L7, a ribosomal housekeeping gene. In addition, exons 4 through 8 of the p53 gene as well as exon 2 of the p21WAF1/CIP1 gene were assayed for mutations by polymerase chain reaction/single stranded conformation polymorphism analysis (PCR/SSCP). Candidate mutations were verified by sequencing. p21WAF1/CIP1/L7 expression was significantly decreased in invasive lesions compared to superficial lesions (P<0.002). p53 mutations were detected by PCR/SSCP in seven tumors [25%] (one superficial, six invasive) and p21WAF1/CIP1/L7 expression was significantly decreased in all tumors that had p53 mutations (P<0.007). PCR/SSCP analysis of exon 2 in p21WAF1/CIP1 detected band shifts in four/28 tumor specimens (two superficial, two invasive), which sequencing and comparison to autologous normal matched DNA revealed as novel mutations.

PTCH gene mutations in invasive transitional cell carcinoma of the bladder.

LOH analysis suggests that multiple tumor suppressor genes play a role in the development of human TCC. The human homolog of the Drosophila PTCH was recently cloned and mapped to the BCNS locus on 9q22.3, a chromosomal region commonly deleted in TCCs. We first examined the steady state mRNA transcription of the PTCH, SMOH and GLI3 genes of the HH signal transduction pathway in TCC cell lines and normal urothelium. Normal urothelium and TCC cell lines express these three genes within the PTCH signal transduction pathway. We then screened for PTCH mutations in 'hot spot' exons 6, 8, 13 and 16 by PCR/SSCP analysis of genomic DNAs from 54 TCC tumor samples and control autologous peripheral blood lymphocytes. DNA sequence analysis confirmed TCC-specific mutations in two of 54 patients (3.7%). These mutations resulted a single amino acid substitution and two frame shifts. One tumor had PTCH mutations in exon 16 as well as exon 13 and one tumor had a mutation in exon 13 alone. Both TCC tumors that contained PTCH mutations had a loss of heterozygosity at 9q. Although the PTCH protein has an unknown function in urothelial cells, the detection of the PTCH, SMOH and GLI3 transcripts in normal urothelium and TCC cell lines and rare PTCH mutations in tumor samples suggest that the HH pathway may have a role in controlling the proliferation of urothelial cells and that PTCH mutations may contribute to the development of a subset of TCCs.

Prostate-specific antigen failure despite pathologically organ-confined and margin-negative prostate cancer: the basis for an adjuvant therapy trial.

PURPOSE: A multivariable analysis to evaluate the potential clinical and pathologic factors that predict for early biochemical failure in patients with pathologically organ-confined and margin-negative disease was performed to define patients who may benefit from adjuvant therapy. PATIENTS AND METHODS: Three hundred forty-one prostate cancer patients treated with a radical retropubic prostatectomy between January 1989 and June 1995 and found to have pathologically organ-confined and margin-negative disease comprised the study population. A logistic regression multivariable analysis to evaluate the predictive value of the preoperative prostate-specific antigen (PSA) level, pathologic (prostatectomy) Gleason score, and pathologic stage on PSA failure occurring during the first postoperative year was performed. RESULTS: Predictors of PSA failure during the first postoperative year in patients with pathologically organ-confined disease included pathologic Gleason score > or = 7 (P = .0007) and preoperative PSA level greater than 10 (P < .0001). Corresponding 3-year freedom-from-PSA-failure rates for these pathologic organ-confined patients with both, one, or neither of these factors were 60%, 75% to 84%, and 95%, respectively (P < .0001). CONCLUSION: Prostate cancer patients with pathologically organ-confined and margin-negative disease and a preoperative PSA level greater than 10 ng/mL or a pathologic Gleason score > or = 7 have significant decrements in short-term PSA-failure-free survival. Therefore, these patients should be considered for adjuvant therapy in the setting of a phase III clinical trial.