Concurrent in situ analysis of point mutations and immune infiltrate in FFPE cancers

Existing methodology for analysis of genetic heterogeneity generally involves digestion of the tumor tissue, followed by bulk DNA extraction or single cell preparation. Such methods destroy the tissue morphology, and therefore opportunities to analyze tumor heterogeneity and clonal architecture within the native spatial context are lost. Thus, there is a clear need for the development of generally applicable methods of in situ mutation detection (ISMD), in which tumor cells can be genetically analyzed in the context of their cellular microenvironment, including immune infiltrate. Furthermore, protocols in which ISMD can be combined with immunohistochemical analysis are highly sought after, as the combination of these two techniques enables insight not only into genetic heterogeneity, but is also permissive of genotype-phenotype analysis, whilst preserving tissue morphology and spatial context. Here we describe a novel method for in situ point mutation detection using commercially available BaseScope reagents, followed by immunohistochemical detection of immune infiltrate on the same tissue section.