How is Microsatellite Instability (MSI) Tested?

Microsatellite Instability (MSI) testing plays a crucial role in identifying tumors with defective DNA mismatch repair (MMR). This is particularly significant in colorectal and endometrial cancers, where MSI status influences prognosis and treatment decisions, including the use of immunotherapy. There are three main methodologies for MSI testing: Immunohistochemistry (IHC), Polymerase Chain Reaction (PCR), and Next-Generation Sequencing (NGS). Each of these techniques has its advantages and limitations, which are discussed in detail below.

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PCR-based MSI testing detects frameshift mutations in coding regions of microsatellites, which is a hallmark of microsatellite instability (MSI-High, MSI-H) tumors. MSI-H tumors arise due to defects in the DNA mismatch repair (MMR) system, leading to instability in microsatellite sequences.

PCR is capable of directly detecting these mutations by analyzing changes in the length of microsatellite repeats.

This method provides a molecular-level assessment of MSI rather than relying on indirect protein expression (as in IHC).

PCR is capable of directly detecting these mutations by analyzing changes in the length of microsatellite repeats.

This method provides a molecular-level assessment of MSI rather than relying on indirect protein expression (as in IHC).

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MSI- Testing

Why is MSI Testing Important?

MSI testing is essential for:

Identifying patients with Lynch Syndrome, a hereditary cancer syndrome.
Determining prognosis in colorectal and endometrial cancers.
Guiding immunotherapy decisions, as MSI-high (MSI-H) tumors respond well to PD-1/PD-L1 inhibitors.

MSI Testing Techniques

There are three commonly used techniques for MSI testing:

1. Immunohistochemistry (IHC)- Detects MMR protein loss.

2. Polymerase Chain Reaction (PCR)- Identifies MSI in microsatellite regions.

3. Next-Generation Sequencing (NGS) – Provides a comprehensive analysis.

Comparison of MSI Testing Techniques

1. Immunohistochemistry (IHC) for MSI Testing

How It Works

IHC detects the loss of MMR protein expression (MLH1, MSH2, MSH6, PMS2) in tumor cells.

If any of these proteins are absent, the tumor is considered MMR-deficient (dMMR), suggesting MSI-H status.

Advantages of IHC

Fast and widely available.

Less expensive than molecular methods.

Helps pinpoint which MMR gene is affected.

Limitations of IHC

Cannot detect MSI directly (only protein loss).

Some MMR mutations still produce a detectable (but non-functional) protein.

Subjective interpretation may lead to errors.

2. Polymerase Chain Reaction (PCR)-Based MSI Testing

How It Works

PCR targets specific microsatellite markers (BAT25, BAT26, NR21, NR24, NR27).

Tumors are classified as MSI-High (MSI-H) if instability is found in â‰¥2 markers.

Advantages of PCR

Directly detects MSI by analyzing DNA sequences.

More sensitive and specific than IHC.

Can identify frameshift mutations in microsatellites.

Limitations of PCR

More expensive than IHC.

Requires tumor DNA and normal DNA for comparison.

Does not provide information on which MMR gene is mutated.

3. Next-Generation Sequencing (NGS) for MSI Testing

How It Works

NGS examines hundreds of microsatellite loci, providing a comprehensive MSI profile.

It can also detect mutations in MMR genes and additional genomic changes.

Advantages of NGS

Simultaneous detection of MSI and MMR mutations.

Higher sensitivity compared to PCR and IHC.

Can assess tumor mutational burden (TMB) for immunotherapy selection.

Limitations of NGS

High cost and longer turnaround time.

Requires specialized bioinformatics analysis.

Not available in all diagnostic labs.

MSI Testing Decision Algorithm

When should MSI testing be performed?

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Scenario	Recommended Testing
Colorectal or endometrial cancer diagnosis	Start with IHC or PCR
Suspicion of Lynch Syndrome	Perform PCR and NGS for confirmation
Unexplained dMMR tumor by IHC	Use NGS to detect somatic mutations
Immunotherapy selection	Use NGS for MSI and TMB status

Clinical Implications of MSI Testing

1. MSI-H Tumors and Immunotherapy

MSI-H tumors respond well to PD-1/PD-L1 inhibitors (e.g., pembrolizumab, nivolumab).

These tumors are often hypermutated, making them more visible to the immune system.

2. Prognostic Importance

MSI-H colorectal cancers have a better prognosis than microsatellite-stable (MSS) cancers.

MSI-H tumors are typically right-sided and have a mucinous or medullary morphology.

3. Differentiating Lynch Syndrome from Sporadic MSI-H Tumors

Feature	Lynch Syndrome	Sporadic MSI-H Cancer
MMR Mutation Type	Germline (inherited)	Somatic (acquired)
MLH1 Promoter Hypermethylation	Absent	Present
Family History of Cancer	Common	Rare
BRAF Mutation Presence	Absent	Common

Conclusion

IHC is a quick and cost-effective initial test.

PCR provides direct MSI detection.

NGS offers the most comprehensive analysis, especially for immunotherapy selection.

By integrating these methodologies, clinicians can identify Lynch Syndrome, guide immunotherapy decisions, and improve patient outcomes.