Dea Nagy

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.

102. Catheter-Based Intrahepatic rAAV Gene Delivery: Influence of Infusion Flow Rate and Volume on Uptake

Catheter-based, fluoroscopy-aided interventional radiology approaches have been utilized in a variety of clinical applications. Safe, consistent and efficient methods for liver directed gene delivery need to be developed. In this study, we examined the effects of infusion flow rate and infusion volume on intrahepatic catheter-based delivery of recombinant adeno-assocated virus 2 vectors (rAAV2) in dogs. To study the efficiency of intrahepatic gene transfer, vector (rAAV-lac Z (nonfunctional promoter)) was delivered through the hepatic artery with an occlusion balloon catheter with a protocol mimicking the clinical angiography procedure of an ongoing clinical gene transfer study in hemophilia B subjects. Three groups of beagle dogs (n=5/group) utilizing three different intrahepatic injection parameters were utilized: A) 8 ml injected at 0.1 ml/min/kg (most similar to ongoing clinical trial), B) 8 ml injected at 8 ml/min/kg and C) 40 ml injected at 8 ml/min/kg. Reproducibility and extent of gene transfer was studied by determining vector distribution and vector copy number in the liver, including individual lobes, 4 weeks post injection. Quantitative PCR using linearized standards was employed to examine vector levels in the liver. Liver histology, serum chemistries and hematology parameters were also examined in all animals over the course of 4 weeks to determine the safety of the various delivery parameters. The results indicate that intrahepatic transgene delivery was influenced by the infusion rate. On average, the rAAV2 copy number per hepatocyte with fast infusion was twice that of slow infusion (e.g. 2.8 copy/cell group B vs 1.4 copy/cell group A). Likewise, vector copy numbers per liver were approximately two times higher in the fast infusion groups (p=0.04, one-way ANOVA). Groups B (higher rate) and C (higher rate and higher volume) did not significantly differ. AAV2 vector tended to be more evenly distributed in individual livers in the fast infusion groups. No changes in chemistries nor hematology values were detected in any dogs over the course of study. Histological analysis of the liver for capsid immunoflourescence is ongoing. This study demonstrates that the infusion flow rate, in particular, can be an important variable in efficient and safe hepatic delivery of AAV.