Evaluation of Polymeric Nanomedicines Targeted to PSMA: Effect of Ligand on Targeting Efficiency.

Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA-). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle.

Autophagic flux determines cell death and survival in response to Apo2L/TRAIL (dulanermin).

BACKGROUND: Macroautophagy is a catabolic process that can mediate cell death or survival. Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment (TR) is known to induce autophagy. Here we investigated whether SQSTM1/p62 (p62) overexpression, as a marker of autophagic flux, was related to aggressiveness of human prostate cancer (PCa) and whether autophagy regulated the treatment response in sensitive but not resistant PCa cell lines. METHODS: Immunostaining and immunoblotting analyses of the autophagic markers p62 [in PCa tissue microarrays (TMAs) and PCa cell lines] and LC3 (in PCa cell lines), transmission electron microscopy, and GFP-mCherry-LC3 were used to study autophagy induction and flux. The effect of autophagy inhibition using pharmacologic (3-methyladenine and chloroquine) and genetic [(short hairpin (sh)-mediated knock-down of ATG7 and LAMP2) and small interfering (si)RNA-mediated BECN1 knock-down] approaches on TR-induced cell death was assessed by clonogenic survival, sub-G1 DNA content, and annexinV/PI staining by flow cytometry. Caspase-8 activation was determined by immunoblotting. RESULTS: We found that increased cytoplasmic expression of p62 was associated with high-grade PCa, indicating that autophagy signaling might be important for survival in high-grade tumors. TR-resistant cells exhibited high autophagic flux, with more efficient clearance of p62-aggregates in four TR-resistant PCa cell lines: C4-2, LNCaP, DU145, and CWRv22.1. In contrast, autophagic flux was low in TR-sensitive PC3 cells, leading to accumulation of p62-aggregates. Pharmacologic (chloroquine or 3-methyladenine) and genetic (shATG7 or shLAMP2) inhibition of autophagy led to cell death in TR-resistant C4-2 cells. shATG7-expressing PC3 cells, were less sensitive to TR-induced cell death whereas those shLAMP2-expressing were as sensitive as shControl-expressing PC3 cells. Inhibition of autophagic flux using chloroquine prevented clearance of p62 aggregates, leading to caspase-8 activation and cell death in C4-2 cells. In PC3 cells, inhibition of autophagy induction prevented p62 accumulation and hence caspase-8 activation. CONCLUSIONS: We show that p62 overexpression correlates with advanced stage human PCa. Pharmacologic and genetic inhibition of autophagy in PCa cell lines indicate that autophagic flux can determine the cellular response to TR by regulating caspase-8 activation. Thus, combining various autophagic inhibitors may have a differential impact on TR-induced cell death.

Improving the biodistribution of PSMA-targeting tracers with a highly negatively charged linker.

BACKGROUND: Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer and in tumor vasculature. Small molecule based inhibitors of PSMA have promised to provide sensitive detection of primary and metastatic prostate tumors. Although significant progress has been made, many of the radiolabeled imaging agents exhibit non-specific background binding. Prevailing tracer designs focus on high affinity urea-based inhibitors with strategically placed hydrophobic patches that interact favorably with the substrate tunnel of PSMA. We hypothesized that a novel PSMA inhibitor design incorporating highly negatively charged linkers may minimize non-specific binding and decrease overall background. METHODS: Through iterative redesign, we generated a series of PSMA inhibitors with highly negatively charged linkers that connect to urea inhibitors and bulky radionuclide chelates. We then performed in vivo imaging and biodistribution studies with the radiolabeled tracers. RESULTS: The tracers derived from our iterative redesign have affinities for PSMA comparable to the "parent" urea ligand Cys-C(O)-Glu. Using a fluorine-18 labeled PSMA targeting tracer, we found that these highly negatively charged molecules exhibit rapid renal excretion with minimal non-specific binding. The biodistribution data at 2 hr showed 4.6%ID/g PC3-PIP tumor uptake with spleen, liver, bone, and blood background levels of 0.1%, 0.17%, 0.1%, and 0.04%, respectively. CONCLUSION: Placement of multiple negative charges in the linker region of PSMA tracers significantly reduced the non-specific background binding without significant reduction of binding affinity. This increased tumor/background contrast in positron emission tomography promises to provide more sensitive tumor detection while decreasing the overall radiation exposure to patients.

A novel PSMA-targeting tracer with highly negatively charged linker demonstrates decreased salivary gland uptake in mice compared to [68Ga]Ga-PSMA-11.

BACKGROUND: The current generation of radiolabeled PSMA-targeting therapeutic agents is limited by prominent salivary gland binding, which results in dose-limiting xerostomia from radiation exposure. JB-1498 is a urea-based small molecule with a highly negatively charged linker targeting prostate specific membrane antigen (PSMA). Prior work on a similar tracer with the same negatively charged linker demonstrated low normal organ/soft tissue background uptake compared to [68Ga]Ga-PSMA-11. The purpose of this study was to investigate if [68Ga]Ga-JB-1498 had reduced salivary gland uptake in mice compared to [68Ga]Ga-PSMA-11. RESULTS: JB-1498 demonstrated high affinity for PSMA binding and tumor uptake in a murine tumor model. In an initial biodistribution study with low molar activity, [68Ga]Ga-JB-1498 demonstrated salivary gland uptake of 0.13 ± 0.01%ID/g. In a second biodistribution study in non-tumor-bearing mice with high molar activity, [68Ga]Ga-JB1498 demonstrated salivary gland uptake of 0.39 ± 0.24% ID/g and kidney activity of 10.12 ± 1.73% ID/g at one hour post IV injection. This salivary gland uptake is significantly less than the published uptake of [68Ga]Ga-PSMA-11. Micro-PET visually confirmed the findings of the biodistribution studies. Dynamic micro-PET imaging demonstrated gradually decreasing [68Ga]Ga-JB1498 activity in salivary glands and kidneys, compared to gradually increasing [68Ga]Ga-PSMA-11 activity in these two organs during the first hour. CONCLUSION: Biodistribution and micro-PET imaging of [68Ga]Ga-JB-1498 demonstrate significantly decreased salivary gland uptake and different pharmacokinetic behavior in kidneys and salivary glands in mice compared to [68Ga]Ga-PSMA-11. Our findings suggest that constructing a PSMA-targeting molecule with a highly negatively charged linker is a promising strategy to reduce salivary gland uptake of GCP-II/PSMA ligands in theranostic applications.

Resveratrol regulates the PTEN/AKT pathway through androgen receptor-dependent and -independent mechanisms in prostate cancer cell lines.

The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) and the androgen receptor (AR) play important roles in tumor development and progression in prostate carcinogenesis. Among many functions, PTEN negatively regulates the cytoplasmic phosphatidylinositol-3-kinase/AKT anti-apoptotic pathway; and nuclear PTEN affects the cell cycle by also negatively regulating the MAPK pathway via cyclin D. Decreased PTEN expression is correlated with prostate cancer progression. Over-expression of AR and upregulation of AR transcriptional activity are often observed in the later stages of prostate cancer. Recent studies indicate that PTEN regulates AR activity and stability. However, the mechanism of how AR regulates PTEN has never been studied. Furthermore, resveratrol, a phytoalexin enriched in red grapes, strawberries and peanuts, has been shown to inhibit AR transcriptional activity in prostate cancer cells. In this study, we use prostate cancer cell lines to test the hypothesis that resveratrol inhibits cellular proliferation in both AR-dependent and -independent mechanisms. We show that resveratrol inhibits AR transcriptional activity in both androgen-dependent and -independent prostate cancer cells. Additionally, resveratrol stimulates PTEN expression through AR inhibition. In contrast, resveratrol directly binds epidermal growth factor receptor (EGFR) rapidly inhibiting EGFR phosphorylation, resulting in decreased AKT phosphorylation, in an AR-independent manner. Thus, resveratrol may act as potential adjunctive treatment for late-stage hormone refractory prostate cancer. More importantly, for the first time, our study demonstrates the mechanism by which AR regulates PTEN expression at the transcription level, indicating the direct link between a nuclear receptor and the PI3K/AKT pathway.

In vivo targeted contrast enhanced micro-ultrasound to measure intratumor perfusion and vascular endothelial growth factor receptor 2 expression in a mouse orthotopic bladder cancer model.

PURPOSE: We evaluated the feasibility of using targeted contrast enhanced micro-ultrasound imaging to assess intratumor perfusion and vascular endothelial growth factor receptor 2 expressions in a mouse orthotopic bladder cancer model. MATERIALS AND METHODS: We created an orthotopic mouse model by implanting MBT-2 murine bladder cancer cell lines in the bladder of syngeneic C3H/He mice (Jackson Laboratory, Bar Harbor, Maine). Successful tumor implantation was confirmed by transabdominal micro-ultrasound imaging on post-implantation day 11. Contrast enhanced micro-ultrasound imaging was done on days 14 and 21. Vascular endothelial growth factor receptor 2 targeted contrast agent was prepared by adding biotinylated anti-vascular endothelial growth factor receptor 2 monoclonal antibodies to streptavidin coated microbubbles. The targeted contrast agents were injected via the retro-orbital route. We quantified intratumor perfusion, vascular endothelial growth factor receptor 2 endothelial expression and blood volume in real time. RESULTS: In the initial study intratumor perfusion data and vascular endothelial growth factor receptor 2 expression could only be measured in 10 of 14 mice (71%) due to motion artifact. We modified our technique by applying an elastic band over the lower abdomen to minimize body wall movement. After the modification complete images were acquired in all mice at 2 consecutive imaging sessions. Measurements were made of intratumor perfusion and in vivo vascular endothelial growth factor receptor 2 expression. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. CONCLUSIONS: Targeted contrast enhanced micro-ultrasound imaging enables investigators to detect and monitor vascular changes in orthotopic bladder tumors. It may be useful for direct, noninvasive, in vivo evaluation of novel anti-angiogenesis therapeutic agents. With the modified technique target enhanced contrast ultrasound can be applied in an orthotopic bladder cancer model.

Targeted, activatable, in vivo fluorescence imaging of prostate-specific membrane antigen (PSMA) positive tumors using the quenched humanized J591 antibody-indocyanine green (ICG) conjugate.

In patients with prostate cancer, a positive surgical margin is associated with an increased risk of cancer recurrence and poorer outcome, yet margin status cannot be determined during the surgery. An in vivo optical imaging probe that could identify the tumor margins during surgery could result in improved outcomes. The design of such a probe focuses on a highly specific targeting moiety and a near-infrared (NIR) fluorophore that is activated only when bound to the tumor. In this study, we successfully synthesized an activatable monoclonal antibody-fluorophore conjugate consisting of a humanized anti-Prostate-Specific Membrane Antigen (PSMA) antibody (J591) linked to an indocyanine green (ICG) derivative. Prior to binding to PSMA and cellular internalization, the conjugate yielded little light; however, after binding an 18-fold activation was observed permitting the specific detection of PSMA+ tumors up to 10 days after injection of a low dose (0.25 mg/kg) of the reagent. This agent demonstrates promise as a method to image the extent of prostate cancer in vivo and could assist with real-time resection of extracapsular extension of tumor and positive lymph nodes.

Optimizing orthotopic bladder tumor implantation in a syngeneic mouse model.

PURPOSE: We established a reliable technique for orthotopically implanting bladder tumor cells in a syngeneic mouse model. MATERIALS AND METHODS: MBT-2 murine bladder cancer cells were transurethrally implanted in the bladder of syngeneic C3H/He mice (Jackson Laboratory, Bar Harbor, Maine). Different chemical pretreatments were used before tumor implantation, including phosphate buffered saline (control), HCl, trypsin and poly-L-lysine. MBT-2 cells (1 x 10(6) or 2 x 10(6)) were instilled into the intravesical space after chemical pretreatment. Tumor take and bladder tumor volume were determined by micro ultrasound. Bladders were harvested at the end of the study to measure bladder weight and for histopathological examination. RESULTS: Bladder pretreatment with HCl in 5 preparations was discontinued due to significant adverse reactions, resulting in death in 1 mouse, and severe bladder inflammation and hematuria 3 days after pretreatment in 2. Pretreatment with phosphate buffered saline, trypsin and poly-L-lysine in 6 animals each was tolerated well without significant adverse reactions or mortality. The tumor take rate in the control, trypsin and poly-L-lysine pretreatment groups was 33%, 83% and 83%, respectively. The take rate was higher in mice instilled with 2 x 10(6) cells than in those with 1 x 10(6) cells (93% vs 73%, p <0.05). CONCLUSIONS: We report a reliable, feasible method of orthotopically implanting bladder tumor cells into a syngeneic mouse model. Poly-L-lysine and trypsin are useful adjunctive pretreatment agents to improve bladder tumor uptake. This model may be suitable to evaluate treatment paradigms for bladder cancer.

A novel alphavirus vaccine encoding prostate-specific membrane antigen elicits potent cellular and humoral immune responses.

PURPOSE: Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). EXPERIMENTAL DESIGN: Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-gamma enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice-compliant toxicology study was conducted in rabbits. RESULTS: PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 x 10(5) infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 10(2) infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. CONCLUSIONS: PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.

A Perspective on the Evolving Story of PSMA Biology, PSMA-Based Imaging, and Endoradiotherapeutic Strategies.

In this review, we cover the evolution of knowledge on the biology of prostate-specific membrane antigen (PSMA) and its translation to therapy. The usual key to discovery is a realistic model for experimentation and for testing a hypothesis. A realistic model is especially needed in the case of the human prostate, which differs significantly from the prostate of species often used as research models. We will emphasize the genetic characterization of PSMA, the nature of the PSMA protein, and its role as a carboxypeptidase, with differing important substrates and products in different tissues. We give special prominence to the importance of PSMA as a target for imaging and therapy in prostate cancer and its underdeveloped role for imaging and targeting the neovasculature of tumors other than prostate cancer. Lastly, we bring attention to its importance in other nonprostatic tissues.

2-5A ligands--a new concept for the treatment of prostate cancer.

Several potent prostate specific membrane antigen (PSMA) inhibitors have been described recently. We generated a PSMA-specific 2-5A ligand called RBI 1033 by linking 2-5A to the N-acetylaspartylglutamate (NAAG)-based inhibitor ZJ-24. We measured the inhibitory activity of RBI 1033 to the folate hydrolase activity of PSMA. Amazingly, we found that compared to ZJ-24 (IC50 = 53.9 nM), RBI 1033 was more than 10 times more potent (IC50 = 4.78 nM) as a folate hydrolase inhibitor, while SMCC 2-5A lacking the ZJ-24 part, did not show much activity (IC50 = 1974 nM). Also, RBI 1033's affinity to PSMA was found to be 10 times higher than ZJ-24 itself.

Novel role of prostate-specific membrane antigen in suppressing prostate cancer invasiveness.

Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein, is overexpressed in prostate cancer. PSMA is a unique cell surface marker, negatively regulated by androgen and extensively used for imaging of hormone refractory carcinomas and metastatic foci. PSMA is a carboxypeptidase with two important enzymatic functions, namely, folate hydrolase and NAALADase. PSMA also exhibits an endocytic function, in which it spontaneously recycles through endocytic vesicles. PSMA is overexpressed at various stages of prostate cancer, including androgen-sensitive and -independent disease, increased in expression with early relapse after therapy. We have used in vitro invasion assays to explore the possible role of PSMA in the metastasis of prostate cancer cells. Androgen-dependent prostate cancer lines, which express PSMA endogenously (e.g., LNCaP, MDA PCa2b, and CWR22Rv1) are less invasive compared with androgen-independent PC3 or DU145 cells, neither of which expresses PSMA. Ectopic expression of PSMA in PC3 cells reduced the invasiveness of these cells, suggesting that this reduction in the invasion capability of PSMA-expressing cells is due to PSMA expression and not to intrinsic properties of different prostate cancer cell lines. Furthermore, knockdown of PSMA expression increased invasiveness of LNCaP cells by 5-fold. Finally, expression of PSMA mutants lacking carboxypeptidase activity reduced the impact of PSMA expression on invasiveness. Thus, it seems that the enzymatic activity is associated with the effect of PSMA on invasiveness.

Targeted elimination of prostate cancer by genetically directed human T lymphocytes.

The genetic transfer of antigen receptors is a powerful approach to rapidly generate tumor-specific T lymphocytes. Unlike the physiologic T-cell receptor, chimeric antigen receptors (CARs) encompass immunoglobulin variable regions or receptor ligands as their antigen recognition moiety, thus permitting T cells to recognize tumor antigens in the absence of human leukocyte antigen expression. CARs encompassing the CD3zeta chain as their activating domain induce T-cell proliferation in vitro, but limited survival. The requirements for genetically targeted T cells to function in vivo are less well understood. We have, therefore, established animal models to assess the therapeutic efficacy of human peripheral blood T lymphocytes targeted to prostate-specific membrane antigen (PSMA), an antigen expressed in prostate cancer cells and the neovasculature of various solid tumors. In vivo specificity and antitumor activity were assessed in mice bearing established prostate adenocarcinomas, using serum prostate-secreted antigen, magnetic resonance, computed tomography, and bioluminescence imaging to investigate the response to therapy. In three tumor models, orthotopic, s.c., and pulmonary, we show that PSMA-targeted T cells effectively eliminate prostate cancer. Tumor eradication was directly proportional to the in vivo effector-to-tumor cell ratio. Serial imaging further reveals that the T cells must survive for at least 1 week to induce durable remissions. The eradication of xenogeneic tumors in a murine environment shows that the adoptively transferred T cells do not absolutely require in vivo costimulation to function. These results thus provide a strong rationale for undertaking phase I clinical studies to assess PSMA-targeted T cells in patients with metastatic prostate cancer.

Should Low Molecular Weight PSMA Targeted Ligands Get Bigger and Use Albumin Ligands for PSMA Targeting?

Prostate Specific Membrane Antigen (PSMA) is strongly expressed in prostate cancer. Recently a number of low-molecular-weight inhibitors have demonstrated excellent PSMA targeting activity for both imaging as well as Lutecium-177 radiotherapy in human trials. The paper by Choy et al raises the question of whether we can further increase the effectiveness of PSMA targeted therapy by adding an albumin-binding entity to low-molecular-weight agents.

Radiolabeled small-molecule ligands for prostate-specific membrane antigen: in vivo imaging in experimental models of prostate cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA) is a cell surface protein that is overexpressed in prostate cancer, including hormone-refractory and metastatic disease. Our goal in this study was to develop a series of PSMA-based imaging agents for clinical use. EXPERIMENTAL DESIGN: We have synthesized and evaluated the in vivo biodistribution of two radiolabeled urea derivatives that have high affinity for PSMA in severe combined immunodeficient mice harboring MCF-7 (breast, PSMA-negative), PC-3 (prostate, PSMA-negative), and LNCaP (prostate, PSMA-positive) xenografts. Radiopharmaceutical binding selectivity and tumor uptake were also evaluated in vivo using dedicated small animal positron emission tomography, single photon emission computed tomography, and gamma scintigraphic imaging devices. N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[(11)C]methyl-L-cysteine ([(11)C]DCMC K(i), 3.1 nmol/L) and N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-[(125)I]iodo-L-tyrosine ([(125)C]DCIT K(i), 1.5 nmol/L) were synthesized using [(11)C]CH(3)I and with [(125)I]NaI/Iodogen, respectively. RESULTS: At 30 minutes postinjection, [(11)C]DCMC and [(125)I]DCIT showed tumor/muscle ratios of 10.8 and 4.7, respectively, with clear delineation of LNCaP-derived tumors on imaging. MCF-7- and PC-3-derived tumors showed significantly less uptake of [(11)C]DCMC or [(125)I]DCIT. CONCLUSION: These results show the feasibility of imaging PSMA-positive prostate cancer using low molecular weight agents.

Selenoprotein P neutralizes lipopolysaccharide and participates in hepatic cell endoplasmic reticulum stress response.

Low serum selenium or selenoprotein P (SePP) levels have been repetitively observed in severe sepsis. The role of SePP in sepsis is incompletely characterized. To test the hypothesis that lipopolysaccharide (LPS) interacts with SePP, we investigated the interaction between LPS and the histidine-rich (His-rich) regions of SePP. We demonstrate that both purified SePP and synthetic peptides corresponding to the His-rich motifs neutralized LPS. In addition, we used a hepatocyte model to study the fate of SePP in response to LPS or endoplasmic reticulum (ER) stress. Our findings indicate that ER stress increases the cellular level of SePP and promotes its nuclear localization.

Preoperative combined nested reverse transcriptase polymerase chain reaction for prostate-specific antigen and prostate-specific membrane antigen does not correlate with pathologic stage or biochemical failure in patients with localized prostate cancer undergoing radical prostatectomy.

PURPOSE: We report a prospective study examining the ability of preoperative nested reverse transcriptase polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) to predict pathologic stage and biochemical recurrence in patients with clinically localized prostate cancer treated with radical prostatectomy. PATIENTS AND METHODS: One hundred forty-one patients were entered onto the study. Preoperative evaluation included clinical T stage, serum PSA, biopsy Gleason score, and serum RT-PCR for PSA/PSM. Univariate and multivariate logistic regression models, Kaplan-Meier estimates, and Cox proportional hazards modeling were used to identify predictors of pathologic stage and biochemical failure. RESULTS: Seventy-three patients (51.8%) were RT-PCR positive for PSA, PSM, or both. In the multivariate logistic regression model, only initial PSA was an independent predictor of pathologic stage as defined by organ-confined disease (odds ratio [OR], 1.06; 95% confidence interval [CI], 1.00 to 1.13; P =.026) or organ-/specimen-confined disease (OR, 1.09; 95% CI, 1.02 to 1.16; P =.009). Overall Kaplan-Meier biochemical relapse-free survival (bRFS) was 85% at 59 months. Multivariate analysis of predictors for bRFS with the Cox proportional hazards model indicated that only initial PSA (OR, 1.05; 95% CI, 1.02 to 1.09; P =.004) and biopsy Gleason score (OR, 3.57; 95% CI, 1.37 to 9.58; P =.009) were independent predictors of biochemical failure. RT-PCR status did not predict pathologic stage or biochemical failure. Repeat analysis excluding 27 patients who received preoperative androgen-deprivation therapy did not change the results. CONCLUSION: Combined nested RT-PCR for PSA and PSM is not an independent predictor of pathologic stage or biochemical failure in patients with localized prostate cancer undergoing radical prostatectomy. This assay has no clinical utility in this patient population.

Glutamate carboxypeptidase II levels in rodent brain using [125I]DCIT quantitative autoradiography.

The ability to visualize quantitatively glutamate carboxypeptidase II (GCPII) levels in vivo could advance our understanding of its function in health and disease. In the current study, we synthesized and evaluated a radiolabeled (iodine-125) analog of N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-iodo-L-tyrosine (DCIT), a potent antagonist of GCPII activity. We examined the regional distribution of [125I]DCIT binding in the rodent brain using quantitative autoradiography in order to confirm the validity of this radioligand as a marker of GCPII in the brain. The ultimate goal is to develop an imaging agent for assessing GCPII levels in the living brain. The specific binding of [125I]DCIT to rat brain followed a regional distribution consistent with previous studies describing regional brain GCPII gene expression and activity. We found a modest rostrocaudal gradient in which specific binding of [125I]DCIT to GCPII was lowest in cortical regions, with increasing levels of binding in midbrain structures and high levels of binding in hindbrain and brainstem. Autoradiography of [125I]DCIT in GCPII knockout and wild type mouse brain showed a gene-dose dependency confirming the selectivity of this radioligand for GCPII. We propose that [125I]DCIT is a selective radioligand that can be used to quantify brain GCPII levels in vitro using quantitative autoradiography.