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

Abstract

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.

Introduction

Detection of clinically relevant mutations in human cancers is commonly performed using genomic analysis of “bulk” tumor extracts or flow cytometry-sorted single cells, however, these approaches do not preserve tissue morphology. Consequently, the interrelationship between mutant cells and their local environment cannot be studied. Novel methods that enable genetic analysis and concurrent analysis of protein expression to be carried out on intact tissue sections are therefore highly sought after.

In situ mutation detection (ISMD) is a powerful tool for the analysis of genetic heterogeneity that preserves spatial and histopathological context. Broadly, there are two approaches currently used for ISMD in a research setting: first in situ PCR-based approaches that rely upon “rolling-circle” amplification of relevant transcripts to generate large fluorescently-labeled DNA coils (Grundberg et al., 2013; Larsson, Grundberg, Soderberg, & Nilsson, 2010), and second “branched” assays such as the commercially available “BaseScope” (from Advanced Cell Diagnostics, ACD), that rely upon sequential binding of complementary DNA oligos to generate a large signal amplification “tree” (Baker et al., 2017).

Assay development for the detection of point mutations in situ has traditionally been problematic as it requires probes capable of discrimination between sequences that differ by only one nucleotide base. Thus, existing ISMD protocols often require extensive optimization and technical expertise, and this has limited their adoption by the wider research community. Upon the release of BaseScope technology by ACD, we performed rigorous testing of its capacity for in situ detection of point mutations, and we found the BaseScope assay offers excellent sensitivity and specificity, with little or no optimization required by the end user (Baker et al., 2017). The assay relies upon the hybridization of a single pair of DNA oligonucleotide “ZZ” probes across the site of a point mutation in an mRNA transcript. This creates a binding platform for further sequential hybridization of a series of complementary DNA oligos, allowing the formation of a large signal amplification “tree” (Fig. 1). Although BaseScope is a relatively new technology, it has already been successfully applied to the detection of somatic point mutations (Baker et al., 2017) and splice variants (Erben, He, Laeremans, Park, & Buonanno, 2018; Zhu et al., 2018), as well as single nucleotide polymorphisms (SNPs) and insertions/deletions (Baker, unpublished) in FFPE tissue sections.

Multiplexing BaseScope ISMD with the in situ analysis of proteins via immunohistochemistry (IHC) is a particularly appealing technique to study genotype-phenotype/environment relationships. Both ISMD and IHC have the capacity to analyze tumor tissue at single cell level, whilst preserving tissue morphology and spatial context. In situ analysis of point mutations and proteins on the same tissue section offers a unique opportunity to study the regulation of protein expression by genetic mutation, cell-of-origin of mutant transcripts and the relationship between mutant clones and their surrounding environment. It has the further benefit of reducing the number of tissue sections required for research, allowing precious archival samples to be used in the most efficient manner.

BaseScope ISMD and conventional IHC lend themselves to multiplexing as the protocols share many of the same steps, for example tissue fixation, target retrieval and signal amplification/detection. However, combining ISH and IHC on the same tissue section is not always straightforward, as the protease treatment that is an essential part of tissue pretreatment for BaseScope probe hybridization could destroy the antigen required for IHC detection. Therefore, careful optimization and validation of protocols is of critical importance. As ISMD with BaseScope is reliant on the preservation of mRNA (which is inherently more unstable than the protein-based antigens required for IHC), we have had greater success with performing the BaseScope assay before IHC (as depicted in Fig. 1).

In the following protocol we describe the concurrent Fast Red-based detection of the KRAS G12V mutation (using ISMD with BaseScope) and DAB-based detection of CD68 + macrophages (using IHC) in FFPE human colorectal cancers. The data generated using this method can be used to gain insight into the immune response elicited by KRAS G12V mutant tumor cells, however with minor modifications the protocol has numerous and broad applications in studying the role of specific somatic mutations in tumor progression.