Single‐cell immunohistochemical mutation load assay (SCIMLA) using human paraffin‐embedded tissues

Abstract

It would be advantageous to measure mutation load in situ in order to determine the relationship between a high mutation load and increased risk for cancer or other diseases and to evaluate sources of possible mutagen exposure. Previously, in situ mutation detection assays have been plagued with multiple rounds of amplification and high rates of false-positives and false-negatives. The single cell immunohistochemical mutation load assay (SCIMLA) was developed to measure somatic mutation frequency, pattern, and spectrum in normal tissues with a single round of amplification. The P53 gene was utilized as a mutation reporter because of the unusual property that missense mutations often cause P53 protein to accumulate in the cell, allowing the mutant proteins to be detected by immunohistochemical staining. Alternative reporter genes with stabilized mutant proteins may be envisioned. Single cells that stain positively for P53 protein overabundance (red cells) were microdissected from ethanol-fixed and paraffin-embedded tissues. A novel stimulated-PCR (S-PCR) protocol permitted successful amplification of a 1.8-kb segment of the P53 gene (i.e., exons 5-9) in 87% of single mammary cells. Subsequent sequence analysis demonstrated that 35% of the amplified red-stained epithelial cells from normal breast tissue have missense mutations at evolutionarily conserved amino acids. Jackpot mutations, presumably due to clonal expansion, were common. False-positive missense mutations at conserved residues were observed in 3% of the clear cells (i.e., without red stain), presumably due to DNA polymerase error in early PCR cycles. The allele dropout rate was measured at 40% of the amplified cells. SCIMLA is applicable to a variety of tissues, utilizes a single amplification of an endogenous gene, displays mutant cells in situ, and may be adapted to other species.

Integrated one- and two-photon imaging platform reveals clonal expansion as a major driver of mutation load

“…Using the FYDR mice, it is now possible to detect the accumulation of cells harboring rare sequence rearrangements while simultaneously studying the extent to which conditions foster clonal expansion of such mutant cells. Although a few studies have analyzed clonal expansion of mutant cells within tissue sections (28)(29)(30), most other mutation assays require tissue disaggregation followed by analysis of either cultured primary cells or genomic DNA [e.g., RMC, Aprt, Tk, Big Blue, Muta Mouse, Gpt⌬ (31-34)], thus limiting information about the clonal relationship among mutant cells. In addition, being able to detect a single fluorescent cell within an entire pancreas [Ϸ3 ϫ 10 7 cells (35)], makes this imaging technique one of the most sensitive methods for detecting rare DNA sequence changes.…”