ERG overexpression and PTEN status predict capsular penetration in prostate carcinoma.

BACKGROUND: This study examines the combined effect of two common genetic alterations, ERG and PTEN, in prostate carcinoma progression. METHODS: Prostate tissue from 90 patients having unilateral capsular penetrating lesions, and a contra-lateral organ confined second lesion, were examined by immunohistochemistry for the expression of the TMPRSS2:ERG transformation product ERG and the loss of expression of PTEN, a powerful phosphatase inhibiting the PI3 kinase pathway. Multivariate logistic regression was carried out to analyze the data. RESULTS: After adjusting for Gleason score, the odds of having capsular penetration were 5.19 times higher (P = 0.015) for ERG+/PTEN- group as compared to the wild type (ERG-/PTEN+). CONCLUSIONS: This study presents the first evidence that ERG over expression and PTEN deletion is associated with greater risk of capsular penetration. Although further studies are needed, these results have the potential to change clinical assessment for prostate cancer.

Testing mutual exclusivity of ETS rearranged prostate cancer.

Prostate cancer is a clinically heterogeneous and multifocal disease. More than 80% of patients with prostate cancer harbor multiple geographically discrete cancer foci at the time of diagnosis. Emerging data suggest that these foci are molecularly distinct consistent with the hypothesis that they arise as independent clones. One of the strongest arguments is the heterogeneity observed in the status of E26 transformation specific (ETS) rearrangements between discrete tumor foci. The clonal evolution of individual prostate cancer foci based on recent studies demonstrates intertumoral heterogeneity with intratumoral homogeneity. The issue of multifocality and interfocal heterogeneity is important and has not been fully elucidated due to lack of the systematic evaluation of ETS rearrangements in multiple tumor sites. The current study investigates the frequency of multiple gene rearrangements within the same focus and between different cancer foci. Fluorescence in situ hybridization (FISH) assays were designed to detect the four most common recurrent ETS gene rearrangements. In a cohort of 88 men with localized prostate cancer, we found ERG, ETV1, and ETV5 rearrangements in 51% (44/86), 6% (5/85), and 1% (1/86), respectively. None of the cases demonstrated ETV4 rearrangements. Mutual exclusiveness of ETS rearrangements was observed in the majority of cases; however, in six cases, we discovered multiple ETS or 5' fusion partner rearrangements within the same tumor focus. In conclusion, we provide further evidence for prostate cancer tumor heterogeneity with the identification of multiple concurrent gene rearrangements.

Antibody-based detection of ERG rearrangement-positive prostate cancer.

TMPRSS2-ERG gene fusions occur in 50% of prostate cancers and result in the overexpression of a chimeric fusion transcript that encodes a truncated ERG product. Previous attempts to detect truncated ERG products have been hindered by a lack of specific antibodies. Here, we characterize a rabbit anti-ERG monoclonal antibody (clone EPR 3864; Epitomics, Burlingame, CA) using immunoblot analysis on prostate cancer cell lines, synthetic TMPRSS2-ERG constructs, chromatin immunoprecipitation, and immunofluorescence. We correlated ERG protein expression with the presence of ERG gene rearrangements in prostate cancer tissues using a combined immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analysis. We independently evaluated two patient cohorts and observed ERG expression confined to prostate cancer cells and high-grade prostatic intraepithelial neoplasia associated with ERG-positive cancer, as well as vessels and lymphocytes (where ERG has a known biologic role). Image analysis of 131 cases demonstrated nearly 100% sensitivity for detecting ERG rearrangement prostate cancer, with only 2 (1.5%) of 131 cases demonstrating strong ERG protein expression without any known ERG gene fusion. The combined pathology evaluation of 207 patient tumors for ERG protein expression had 95.7% sensitivity and 96.5% specificity for determining ERG rearrangement prostate cancer. In conclusion, this study qualifies a specific anti-ERG antibody and demonstrates exquisite association between ERG gene rearrangement and truncated ERG protein product expression. Given the ease of performing IHC versus FISH, ERG protein expression may be useful for molecularly subtyping prostate cancer based on ERG rearrangement status and suggests clinical utility in prostate needle biopsy evaluation.

Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.

In a clinical study of recombinant adeno-associated virus-2 expressing human factor IX (AAV2-FIX), we detected 2 impediments to long-term gene transfer. First, preexisting anti-AAV neutralizing antibodies (NABs) prevent vector from reaching the target tissue, and second, CD8(+) T-cell responses to hepatocyte-cell surface displayed AAV-capsid-terminated FIX expression after several weeks. Because the vector is incapable of synthesizing viral proteins, a short course of immunosuppression, until AAV capsid is cleared from the transduced cells, may mitigate the host T-cell response, allowing long-term expression of FIX. To evaluate coad-ministration of immunosuppression, we studied AAV8 vector infusion in rhesus macaques, natural hosts for AAV8. We administered AAV8-FIX in 16 macaques via the hepatic artery and assessed the effects of (1) preexisting anti-AAV8 NABs, (2) a standard T-cell immunosuppressive regimen, and (3) efficacy and safety of AAV8-FIX. We found that low titers (1:5) of preexisting NABs abrogate transduction, whereas animals with undetectable NABs are safely and effectively transduced by AAV8-FIX. Coadministration of mycophenolate mofetil and tacrolimus with vector does not induce toxicity and does not impair AAV transduction or FIX synthesis. These findings enable a clinical study to assess the effects of immunomodulation on long-term FIX expression in patients with hemophilia B.

Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs.

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.

Overlesioned hemiparkinsonian non human primate model: correlation between clinical, neurochemical and histochemical changes.

Monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have been widely used as animal models of Parkinson's disease (PD). Depending on the method of administration different PD models can be developed. Systemic (iv, sc.) MPTP administration can induce an advanced parkinsonian syndrome. However, systemic administration may require intensive animal care after neurotoxin administration, as well as repeated high doses of MPTP to avoid spontaneous recovery. Unilateral intracarotid artery (ICA) MPTP administration induces a stable hemiparkinsonian syndrome, with the advantage of allowing the animal to groom and feed itself and having a control side in the same animal. However, this unilateral syndrome lacks the bilateral characteristics of advanced PD. Bilateral ICA administration can induce a reliable bilateral syndrome but inherent is the risk of severely impairing the animals and leaving them unable to maintain themselves. This report analyzed the PD model induced by administration of unilateral ICA and subsequent intravenous injections of MPTP in rhesus monkeys. The combined method of MPTP administration induces an advanced stable parkinsonian syndrome, in which the ICA injection of MPTP initiates the parkinsonian syndrome primarily in one hemisphere and the subsequent iv. doses (administered as needed) further deplete the dopamine (DA) system to induce a bilateral lesion in a shorter period of time, with fewer side effects. We studied the relationships between the behavioral, biochemical and histochemical changes related to the combined MPTP treatments to further characterize this model. The monkeys were categorized as presenting mild (stage 2) or moderate (stage 3) parkinsonism based on a parkinsonian rating scale. Postmortem biochemical analysis showed massive DA reduction equally in the caudate nucleus and putamen ipsilateral to ICA MPTP infusion, with varying degrees of DA preservation in the contralateral striatum. Differences between stage 2 and stage 3 were attributed to DA concentrations in the caudate nucleus and putamen of the contralateral hemisphere. Tyrosine hydroxylase immunohistochemistry revealed that the midbrain DA neurons of the group A8, A9, and A10 showed differential vulnerability for MPTP. This finding was similar to that observed in idiopathic PD with significant relationships between the clinical stages and cell losses in the group A9 (substantia nigra pars compacta). Positron emission tomography (PET) using [18F] 6-fluoro-L-m- tyrosine (FMT) showed that uptake (Ki) values correlated well with the biochemical data and are good predictors of DA levels in the contralateral striatal regions. Consistent with the immunohistochemical analysis, PET data also showed significant correlations with all groups of the DA cells. Here we describe an animal model that can play an important role in understanding the symptoms and therapeutic basis of PD since different severities of parkinsonian symptoms can be mimicked.

Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII.

Using separate adeno-associated viral 2 (AAV2) vectors to deliver the heavy and light chains of factor VIII (FVIII) we have overcome the packaging limitations of AAV, achieving phenotypic correction of hemophilia A in mice. AAV vectors were constructed that use a liver-specific promoter and the cDNA sequences of either the human or canine heavy and light chains of FVIII. After intraportal vein injection of these vectors in hemophilia-A mice, therapeutic to superphysiologic levels of active FVIII were achieved in plasma in a dose-dependent manner. Phenotypic correction of the bleeding diathesis was demonstrated by survival of all treated mice after tail clipping. Biochemical analysis demonstrated lower levels of heavy-chain (25- to 100-fold) compared with light-chain protein in the plasma of treated animals. Differences in gene transfer and transcription did not account for the differences in protein expression. We hypothesize that improvements in FVIII activity could be achieved by improvements in FVIII heavy-chain expression. This work demonstrates that cotransduction of liver with AAV vectors expressing the heavy and light chains of FVIII corrects hemophilia A in vivo, providing an alternative approach to the use of a single vector. This strategy may potentially be useful for other large therapeutic proteins that contain functionally distinct domains.

Sustained phenotypic correction of canine hemophilia A using an adeno-associated viral vector.

Gene therapy for hemophilia A requires efficient delivery of the factor VIII gene and sustained protein expression at circulating levels of at least 1% to 2% of normal. Adeno-associated viral type 2 (AAV2) vectors have a number of advantages over other viral vectors, including an excellent safety profile and persistent gene expression. However, a major disadvantage is their small packaging capacity, which has hampered their use in treating diseases such as hemophilia A, cystic fibrosis, and muscular dystrophy, which are caused by mutations in large genes. Here we demonstrate that this can be overcome by using small regulatory elements to drive expression of a B-domain-deleted form of FVIII. The use of this vector for hepatic gene transfer in a canine model of hemophilia A resulted in the sustained (> 14 months) expression of biologically active FVIII. FVIII activity levels of 2% to 4% were achieved. These levels correlated with a partial correction in the whole-blood clotting time and cuticle bleeding time. In addition, immunoprecipitation analysis demonstrated the expression of canine FVIII of the predicted size in the plasma of injected animals. These data support the use of AAV2 vectors in human clinical trials to treat hemophilia A patients.