Automated multiplexing quantum dots in situ hybridization assay for simultaneous detection of ERG and PTEN gene status in prostate cancer

The photostability and narrow emission spectra of nonorganic quantum dot fluorophores make them desirable detection methods for ultrasensitive and multiplexing in situ hybridization applications to identify genetic aberrances in morphologically preserved clinical tissue specimens. However, robustness and reliability have not been fully investigated for quantum dot fluorophores in situ hybridization applications. We demonstrate the feasibility of an automated multiplexing four-color quantum dot fluorophores in situ hybridization assay comprised of four genomic probes each labeled with unique haptens, four anti-hapten antibodies each conjugated with quantum dot fluorophores with distinct emission spectrum, protocols for their use on a fully automated tissue staining platform, and direct observation of multiple signals using conventional filter-based fluorescent microscopy. This assay is successfully applied to the simultaneous detection of ERG3p, ERG5p, PTEN, and CEN10 genes in formalin-fixed, paraffin-embedded prostate tissues on BenchMark ULTRA instruments. There were 386 slides from 10 prostatectomy cases stained on 13 on these instruments. These 10 cases consisted of benign prostate and prostate cancer; the cancer cases were either positive or negative for ERG rearrangement and/or contained PTEN deletion. There were 350 (91%) slides appropriately stained for all four targets. The staining results accurately identified the ERG and PTEN status for all 10 cases. This approach is expected to enable multiplexing in situ detection of molecular biomarkers in routinely processed human clinical specimens.