Automated quantum dots ISH assay for detection of ERG rearrangement and PTEN deletion in prostate cancer.

Abstract

5045 Background: ERG rearrangement and PTEN deletion are the two most common genomic events in prostate cancer. Overexpression of the ERG protein caused by rearrangement of the ERG gene has been frequently associated with more aggressive prostate cancers and a poor prognosis. PTEN genomic deletion and absence of PTEN expression are associated with unfavorable clinical outcome. To detect ERG rearrangement and PTEN deletion simultaneously, we developed a four-color multiplex ISH assay using non-organic quantum dots (QD). The photo-stability and narrow emission spectra of QDs makes them desirable for ultrasensitive and multiplexing ISH applications. The automated QD ISH assay allows adequate delivery of QDs to nuclear targets and reproducible detection of ERG and PTEN gene targets. Methods: DNA probes specific for ERG 5’, ERG 3’, PTEN and CEN 10 were labeled with different haptens, digoxigenin (DIG), dinitrophenyl (DNP), thiazole sulfonamide (TS), or nitropyrazole (NP). QDs with 565, 655, 605 and 525nm wavelengths were conjugated to the anti- DIG, DNP, TS or NP antibodies, respectively. The multiplex QD ISH assay was fully automated on the VENTANA BenchMark ULTRA. FFPE prostate tissue slides were hybridized with the labeled probes. The probes were then detected by the QD-conjugated antibodies and visualized under fluorescent microscope. Results: We performed the multiplex QD ISH assay on 386 slides with tissue sections from 10 prostate specimens on 13 BenchMark ULTRA instruments. The 10 cases consisted of either benign prostate tissues or prostate cancer, positive or negative for ERG rearrangement and/or PTEN deletion. 350 (91%) of the slides were successfully stained for all 4 molecular targets. The expected ERG and PTEN status were detected with high reproducibility. Conclusions: We developed an automated QD based multiplex ISH assay to simultaneously detect ERG rearrangement and PTEN deletion in prostate cancer. The assay is highly sensitive and reproducible. It enables investigation of potential clinical use of ERG and PTEN as predictive or prognostic markers. In addition, the same technology is expected to enable multiplex in situ detection of other molecular biomarkers using standard clinical specimens.