Single-Cell Immunohistochemical Mutation Load Assay (SCIMLA) Using Human Paraffin-Embedded Tissues

Authors

Gudrun Schlake, City of Hope National Med Center
Qiang Liu, City of Hope National Med Center
Ernst Heinmöller, City of Hope National Med Center
Kathleen A. Hill, City of Hope National Med Center
Lawrence Weiss, City of Hope National Med Center
Steve S. Sommer, City of Hope National Med Center

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. © 2003 Wiley-Liss, Inc.

Publication Title

Environmental and Molecular Mutagenesis

Recommended Citation

Schlake, G., Liu, Q., Heinmöller, E., Hill, K., Weiss, L., & Sommer, S. (2003). Single-Cell Immunohistochemical Mutation Load Assay (SCIMLA) Using Human Paraffin-Embedded Tissues. Environmental and Molecular Mutagenesis, 42 (3), 206-215. https://doi.org/10.1002/em.10193