158 Background: Clinically, CTCs are used primarily in longitudinal monitoring of metastatic disease progression. However, the analysis of specific CTC biomarkers has the potential to optimize patient management by identifying those likely to respond to specific targeted agents. Often, mCRPC is driven by dysregulation of the androgen receptor (AR) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) oncogenic pathways such that blockade of one pathway stimulates the other. These functional deficits in the AR and PI3K/AKT pathways are associated with fusion of the TMPRSS2 and ERG genes and with loss of PTEN protein, respectively. Therefore, we sought to characterize CTCs for the simultaneous presence of both these biomarkers using immunofluorescence (IF) and fluorescence in situ hybridization (FISH). Methods: Nucleated peripheral blood cells from mCRPC patients were attached to slides and examined using CTC technology (Epic Sciences). Cytokeratin-positive/CD45-negative cells with an intact nucleus and a malignancy-consistent morphology were identified as CTCs, and their exact positions on the slides were recorded. The slides were then subjected to multiplex Quantum Dot IF and FISH procedures with anti-AR, -ERG, and -PTEN antibodies and 5’ERG, 3’ERG, PTEN, and Cen10 probes, respectively, on an automated slide-staining platform (Ventana Medical Systems, Inc.). The IF and FISH signals were visualized by spectral imaging (Ventana). Results: The automated IF/FISH staining procedure facilitated multiplex characterization of individual CTCs from patient samples for the protein biomarker targets AR, ERG, and PTEN and the genomic biomarker targets 5’ERG, 3’ERG, PTEN, and Cen10. Conclusions: This method for high-sensitivity, multiplex molecular characterization of critical CTC biomarkers in mCRPC patients might aid oncologists in identifying and stratifying those patients likely to respond to combination therapy with targeted PI3K/AKT inhibitors and anti-androgens/Cyp17 inhibitors. Patient trials examining the clinical utility of this assay in mCRPC are currently underway.
Virulence Gene Profiling and Pathogenicity Characterization of Non-Typhoidal Salmonella Accounted for Invasive Disease in Humans