FISH Assays

Epic Sciences can identify gene amplifications, deletions and rearrangements by DNA fluorescence in situ hybridization (FISH). This test consists of:

1. Slide prep: Upon receipt of patient blood sample, whole blood is lysed and nucleated cells are deposited on up to 12 microscope slides that are frozen at -80 ºC until analysis.
2. Cell staining and scanning: Slides are immunofluorescently stained and scanned by Epic’s rapid fluorescent scanning method, which images each nucleated cell.
3. CTC identification and biomarker analysis: A digital pathology algorithm, which includes protein expression and morphology, differentiates candidate CTCs from surrounding white blood cells (WBCs). CTCs are confirmed and classified into one of the following categories (1) Traditional CTCs, (2) CTC clusters, (3) CK(-) CTCs and (4) Apoptotic CTCs (see CTC basics to review these different classes of CTCs).
4. FISH Assay: Coverslips are removed from the slides, stain is attenuated, slides are fixed and dehydrated, and lastly, hybridized with FISH probes. CTCs are relocated using Epic’s software and scored. FISH results are compiled in a report with CTC enumeration and biomarker expression data.

The data below shows CTCs that were evaluated using a PTEN FISH assay developed on the Epic platform. PTEN status in CTCs was compared to matched, archived and fresh tumor tissue samples from 41 mCRPC patients. We found that PTEN status in CTCs and fresh tissue were concordant. There was more discordance when comparing CTCs to archived primary tissue. Analysis of gene status in CTCs may be representative of the current disease and could serve as a non-invasive solution for genetic characterization.

Representative images of PTEN and CEP10 genotype in patient CTCs. Top panel shows CTCs that were identified by immunofluorescence and bottom panel shows the PTEN FISH analysis of the same CTCs indentified above.

Epic Sciences has developed numerous assays, which are being used to test more than 10,000 patient blood samples from clinical trial sites located in North America, Europe and Asia. These protein, FISH and genomic assays are designed to measure protein abnormalities in CTCs for 12 different cancer types.