Tumor-specific microsatellite instability: do distinct mechanisms underlie the MSI-L and EMAST phenotypes?

Abstract

Microsatellite DNA sequences display allele length alterations or microsatellite instability (MSI) in tumor tissues, and MSI is used diagnostically for tumor detection and classification. We discuss the known types of tumor-specific MSI patterns and the relevant mechanisms underlying each pattern. Mutation rates of individual microsatellites vary greatly, and the intrinsic DNA features of motif size, sequence, and length contribute to this variation. MSI is used for detecting mismatch repair (MMR)-deficient tumors, which display an MSI-high phenotype due to genome-wide microsatellite destabilization. Because several pathways maintain microsatellite stability, tumors that have undergone other events associated with moderate genome instability may display diagnostic MSI only at specific di- or tetranucleotide markers. We summarize evidence for such alternative MSI forms (A-MSI) in sporadic cancers, also referred to as MSI-low and EMAST. While the existence of A-MSI is not disputed, there is disagreement about the origin and pathologic significance of this phenomenon. Although ambiguities due to PCR methods may be a source, evidence exists for other mechanisms to explain tumor-specific A-MSI. Some portion of A-MSI tumors may result from random mutational events arising during neoplastic cell evolution. However, this mechanism fails to explain the specificity of A-MSI for di- and tetranucleotide instability. We present evidence supporting the alternative argument that some A-MSI tumors arise by a distinct genetic pathway, and give examples of DNA metabolic pathways that, when altered, may be responsible for instability at specific microsatellite motifs. Finally, we suggest that A-MSI in tumors could be molecular signatures of environmental influences and DNA damage. Importantly, A-MSI occurs in several pre-neoplastic inflammatory states, including inflammatory bowel diseases, consistent with a role of oxidative stress in A-MSI. Understanding the biochemical basis of A-MSI tumor phenotypes will advance the development of new diagnostic tools and positively impact the clinical management of individual cancers.

Figure 1. MSI Analysis of MMR+ and MMR- cell lines at the BAT26 and D2S123 loci

PCR reactions were performed using genomic DNA isolated from MMR+ U2OS and MMR-HCT116 or L174T cell lines and primer sets in which one primer was labeled with WellRED D3 dye (BAT26) or WellRED D4 dye (D2S123). The amount of input DNA, MgCl₂ concentration, and annealing temperature were optimized for each primer set. PCR products and size standards were mixed with formamide, run on a Beckman Coulter CEQ 8000 system, and analyzed using the Fragment Analysis software. A. MSI analysis using the BAT26 marker. DNA from U2OS cells shows stability at the quasimonomorphic locus with a fragment of 119nt (PCR stutter bands are also evident). DNA from MMR-deficient HCT116 cells display instability with a shift to a smaller allele size. B. MSI analysis using the D2S123 marker. DNA from U2OS cells is homozygous and stable for this allele, but DNA from MMR⁻ L174T show extra alleles as indicated by the arrows. C. Ambiguity at the D2S123 marker. Amplification of DNA from HCT116 cells showed minor new alleles (arrows with asterisk) that were not resolved upon utilizing a proofreading-proficient DNA polymerase in the PCR reaction.

Figure 2. MSI specificity in tumor cells reflects underlying genetic alterations

The microsatellite markers used to detect tumor-specific MSI are very diverse at the DNA sequence level (see Table 2 for details). In diagnostics, the primary microsatellite markers used to diagnose Lynch Syndrome are mononucleotide repeats (purple circle). Dinucleotide repeats (green circle) and tetranucleotide repeats (blue circle) have been used to detect the MSI-L and EMAST phenotypes, respectively. Loss of the major mismatch repair proteins MSH2 and MLH1 results in widespread destabilized of all microsatellite motifs, and the MSI-H phenotype (red intersect). Alternative MSI (A-MSI) phenotypes arise when other genome maintenance pathways are disrupted. Loss of MSH6 disrupts only MutSα-mediated repair, and destabilizes only mononucleotides, whereas loss of MSH3 disrupts only MutSβ-mediated repair and destabilizes both di- and tetranucleotides. Loss of PMS2 results in a higher rate of instability at dinucleotide repeats. Increased expression of alkyladenine glycosylase (AAG) results in MSI at mono- and dinucleotides. Based on in vitro characterization of DNA polymerase fidelity at various microsatellites, tumors with increased expression of Pol β are expected to display A-MSI restricted to mononucleotides, while tumors with decreased expression of Pol κ are expected to display A-MSI restricted to di- and tetranucleotide repeats. See text for details and references.