Genotyping of phenotypically defined cells in neoplasia: enhanced immunoFISH via tyramide signal amplification (TSA) segregates immunophenotypically-defined cell populations for gated genotyping.

Molecular morphologic tools exist for simultaneously visualizing immunophenotype and genotype of tumors, but are frequently hampered by a delicate balance between removing sufficient amount of the protein blocking full access of the probe to hybridize to target nucleic acids while still preserving sufficient target antigen for immunophenotyping. The result is often suboptimal, with either insufficiently visualized gene deletions and amplifications due to masking protein, or overdigestion of the protein target. Our purpose was to design and validate a gated genotyping assay that enables optimal and concomitant detection of both gene and protein. Using the proliferating endothelial cell compartment within gliomas organized in a tissue microarray (TMA), we tested the hypothesis that tyramide signal amplification (TSA) with deposition of a fluorochrome could be used during immunophenotyping, permitting sufficient protein digestion while insuring probe accessibility to nucleic acid target. The method was successfully validated using a TMA containing 38 glioma cases previously genotyped for EGFR amplification. CD31 positive endothelial cells were segregated via TSA-based Alexa-Fluor 647 immunofluorescence for analysis of EGFR amplification of the gliomas organized in the TMA. Enhanced immunoFISH (TSA) successfully segregates immunophenotypically-defined cell populations for gated genotyping.