Table 1 Comparison of different assays used to detect mismatch repair defects and other predictors of immune therapy response or resistance

MSI-PCR

PCR-based amplification of known microsatellite loci, gel electrophoresis, and software scoring to detect instability as a multiple-band pattern in amplicons

Focused test with rapid turn-around time

Interpretation difficulties, limited to MSI diagnosis, no information on genetic source of MMRd

dMMR/IHC

Antibody-based staining of FFPE sections from tumor, followed by microscopic examination and scoring to detect MMR proteins (MSH2, MSH6, MLH1, PMS2)

Focused test using a conventional pathology approach, inexpensive, rapid turn-around time

Evaluates the end result of MMR protein depleting alterations only, subject to inter-individual interpretation

CIN/FISH

Hybridization of centromere-specific fluorescent probes to chromosomal spreads, microscopic scoring of centromeric counts to detect aneuploidy

Genome-wide evaluation of chromosomal instability

Counting-based evaluation that is subject to inter-individual interpretation variability

MMR/MSI-NGS panel

NGS of genes for MMR proteins, mutation detection and annotation of pathogenic variants

Focused evaluation of mutations across known MMRd genes

Insensitive to large-scale alterations such as CNVs, insufficient breadth for TMB or neoantigen prediction

NGS/WES

NGS of all known coding exons of genes, mutation detection and annotation of pathogenic variants in genes

Unbiased evaluation of mutations across all coding genes, MSI evaluation (added probes), neoantigen prediction, TMB enabled

Mutational signature calculation may be compromised by lack of breadth

NGS/WGS

NGS of whole genome libraries from cancer and matched normal DNA, comprehensive variant detection, and annotation of variants

Unbiased evaluation of mutations across all coding genes, MSI evaluation, neoantigen prediction, TMB, mutational signature enabled

Expensive to generate sufficient coverage from low cellularity tumors