A microsatellite instability test evaluates whether a tumor has accumulated abnormal length changes in short repetitive DNA sequences called microsatellites. These errors usually arise when the DNA mismatch repair system is deficient. An MSI-high result can help classify a tumor, predict benefit from immune checkpoint therapy in appropriate settings, and identify people who may need evaluation for Lynch syndrome. MSI testing does not diagnose Lynch syndrome by itself. Most MSI-high tumors are caused by acquired changes confined to the cancer, while a smaller proportion result from an inherited pathogenic variant in a mismatch repair gene. Laboratories may assess instability by PCR or next-generation sequencing, or evaluate the related mismatch repair proteins by immunohistochemistry. The methods are complementary but not identical, and performance can vary by tumor type. A stable result does not exclude every mismatch repair defect or hereditary cancer syndrome. Interpretation depends on tumor content, specimen quality, assay markers, protein-staining pattern, age, cancer type, family history, and follow-up tests such as MLH1 promoter methylation, BRAF analysis, somatic tumor testing, and germline testing.
- MSI is a tumor phenotype caused most often by deficient DNA mismatch repair.
- MSI-high and deficient mismatch repair are closely related but not perfectly interchangeable results.
- PCR, next-generation sequencing, and mismatch repair immunohistochemistry use different signals.
- MSI-high status can guide immunotherapy in validated cancer and treatment settings.
- An abnormal tumor screen raises concern for Lynch syndrome but does not prove inheritance.
- MLH1 promoter methylation, BRAF testing, paired tumor-normal analysis, and germline testing may clarify cause.
Table of Contents
- Mismatch repair and microsatellite instability
- Why MSI testing is ordered
- PCR, NGS, and immunohistochemistry
- How to read MSI and MMR results
- MSI testing and Lynch syndrome
- Treatment and prognostic implications
- Discordant, negative, and limited results
- Next steps after testing
Mismatch Repair and Microsatellite Instability
DNA is copied each time a cell divides. The copying machinery occasionally inserts the wrong base or slips while copying repetitive sequences. The mismatch repair system detects and corrects many of these errors. Its core proteins include MLH1, PMS2, MSH2, and MSH6, which function as paired complexes. When both functional copies of a mismatch repair gene are lost or silenced in a tumor, errors accumulate.
Microsatellites are short repeated DNA motifs. Because the repeats look similar, DNA polymerase can add or omit repeat units during replication. A functioning mismatch repair system repairs the insertion-deletion loop. A deficient system allows the altered length to persist. When multiple microsatellite markers change length in tumor DNA, the phenotype is called microsatellite instability.
MSI is a consequence of repair deficiency, while deficient mismatch repair, or dMMR, describes the underlying pathway state. They usually agree but measure different things. MSI PCR or NGS evaluates DNA instability. Mismatch repair immunohistochemistry evaluates whether the four major proteins are present in tumor-cell nuclei. A tumor can have intact-looking proteins that are nonfunctional, or it can have an unusual biology in which the tests disagree.
Mismatch repair deficiency can result from:
- inherited and then somatically inactivated variants in MLH1, MSH2, MSH6, or PMS2;
- a deletion involving the 3′ end of EPCAM that causes epigenetic silencing of nearby MSH2;
- two acquired variants in a mismatch repair gene;
- acquired MLH1 promoter methylation;
- less common structural, regulatory, or treatment-related mechanisms.
The high mutation burden generated by dMMR creates abnormal proteins that may be recognized by the immune system. This helps explain why many MSI-high cancers respond to immune checkpoint blockade. However, MSI is not the only determinant of response, and clinical use depends on tumor type, stage, prior therapy, drug indication, and current guidelines [1].
Why MSI Testing Is Ordered
MSI or MMR testing is ordered for two major reasons: to guide cancer management and to screen tumors for possible Lynch syndrome. A single tumor test can serve both purposes, but the follow-up pathways differ.
Universal or broad tumor screening is commonly used in colorectal and endometrial cancers because relying only on young age or family history misses people with Lynch syndrome. Some health systems and professional guidelines also recommend testing in additional cancers or whenever the result would affect treatment. The ordering indication should be stated because a laboratory may choose a different preferred method by tumor type.
For treatment selection, MSI-high or dMMR status may support use of immune checkpoint inhibitors in approved or guideline-supported settings. Testing may be performed on a new diagnosis, recurrence, or metastatic specimen. It can be part of a broad tumor sequencing panel or ordered as a focused biomarker.
For hereditary-risk screening, the goal is not to prove Lynch syndrome but to identify tumors with a repair-deficient pattern that warrants additional evaluation. The pattern of protein loss can direct the next test. Loss of MLH1 and PMS2 often leads to assessment of MLH1 promoter methylation and, in colorectal cancer, sometimes BRAF p.V600E. Loss of MSH2 and MSH6 points toward MSH2 or EPCAM evaluation; isolated MSH6 loss points toward MSH6; isolated PMS2 loss points toward PMS2, while recognizing technical and biological exceptions.
MSI may also contribute to prognosis. In localized colorectal cancer, MSI-high status has established associations with tumor behavior and can affect adjuvant-treatment discussions. Prognostic meaning is stage and treatment dependent and should not be generalized across all cancers.
The test is performed on tumor tissue, not as a stand-alone blood test for Lynch syndrome. Germline testing analyzes constitutional DNA and asks whether an inherited pathogenic variant is present. Tumor MSI asks whether the cancer shows repair failure. Both may be needed, but they are not substitutes.
PCR, NGS, and Immunohistochemistry
PCR-based MSI testing amplifies a panel of microsatellite markers from tumor DNA, often with matched normal DNA or validated monomorphic markers. The laboratory compares fragment lengths. Instability at a sufficient number or proportion of markers produces an MSI-high classification. Traditional panels included dinucleotide and mononucleotide markers; modern mononucleotide panels generally provide stronger performance across many tumors.
Next-generation sequencing detects length variation across microsatellite loci and may calculate an MSI score. MSI can be included in a broad panel that also reports mutations, copy number, fusions, and tumor mutation burden. Different NGS assays use different loci, algorithms, and thresholds. Validation in the relevant tumor types is essential because performance can be lower when tumor content is poor or the cancer has an atypical instability pattern.
Mismatch repair immunohistochemistry stains tissue for MLH1, PMS2, MSH2, and MSH6 proteins. Normal internal cells—such as lymphocytes, stromal cells, or benign glands—should retain nuclear staining and serve as controls. Loss means tumor nuclei lack convincing staining while internal controls remain positive. The paired biology helps interpret patterns: MLH1 loss commonly causes secondary PMS2 loss, and MSH2 loss commonly causes secondary MSH6 loss.
| Method | Main output | Important advantage | Important limitation |
|---|---|---|---|
| MSI PCR | MSI-high, stable, and sometimes MSI-low | Directly measures instability; well established | Marker performance can vary by tumor type; needs adequate tumor DNA |
| MSI by NGS | Algorithmic MSI score or classification | Can be bundled with broad genomic profiling | Platform-specific loci and cutoffs; low tumor fraction can cause false negatives |
| MMR immunohistochemistry | Retained or lost MLH1, PMS2, MSH2, MSH6 expression | Indicates which gene pathway may be affected and preserves tissue context | Missense variants may retain staining; fixation and interpretation can cause error |
The College of American Pathologists guideline recommends selecting among methods according to cancer type and treatment purpose rather than assuming one method is best for every tumor [1]. For example, MMR immunohistochemistry may be preferred in some endometrial-cancer contexts, while PCR or NGS can be appropriate in others.
Tumor enrichment matters. A pathologist marks viable tumor and estimates cellularity. Necrosis, mucin, inflammation, and treatment effect can reduce tumor DNA. For immunohistochemistry, poor fixation may produce weak or patchy staining. Repeating the stain on another block or using molecular MSI testing may resolve an equivocal result.
How to Read MSI and MMR Results
An MSI report may use three categories:
- MSI-high: instability is detected at or above the assay threshold.
- Microsatellite stable, or MSS: no qualifying instability is detected.
- MSI-low: instability is detected below the high threshold, a category used by some PCR assays.
Many current clinical decisions group MSI-low with MSS, but the exact report and disease context should be reviewed. NGS assays may use “MSI indeterminate” when the score is near the cutoff or too few loci are evaluable.
An MMR immunohistochemistry report may say proficient MMR, or pMMR, when all four proteins are retained, and deficient MMR, or dMMR, when one or more are lost. The pattern guides but does not definitively identify the causal gene.
| IHC pattern | Common implication | Typical clarification |
|---|---|---|
| MLH1 and PMS2 lost | MLH1 inactivation, often acquired methylation | MLH1 promoter methylation; colorectal tumors may also use BRAF analysis |
| MSH2 and MSH6 lost | MSH2 pathway inactivation | Germline and/or tumor analysis of MSH2 and EPCAM |
| Isolated MSH6 loss | MSH6 inactivation | MSH6 germline and somatic evaluation |
| Isolated PMS2 loss | PMS2 inactivation, or occasionally MLH1 alteration | PMS2-focused evaluation with attention to MLH1 mechanisms |
| All proteins retained | pMMR by IHC | MSI testing may still be used if suspicion or treatment context warrants |
A result should be called abnormal only when internal controls are adequate. “Equivocal,” “subclonal,” or “heterogeneous” loss requires expert review. Some tumors show loss only in a portion of the cancer, which may reflect true subclonal inactivation or technical variation.
MSI-high and dMMR are biomarkers, not stage measurements. They do not quantify how much cancer is present. They also do not identify the exact mismatch repair mutation. The full report should state method, specimen, tumor content, markers or algorithm, result, and recommended follow-up.
MSI Testing and Lynch Syndrome
Lynch syndrome is caused by an inherited pathogenic variant affecting mismatch repair. People with Lynch syndrome have increased risks for colorectal, endometrial, and several other cancers, with the risk spectrum varying by gene. Tumors often become dMMR after the remaining working gene copy is inactivated.
An MSI-high or abnormal IHC result raises the possibility of Lynch syndrome, but acquired causes are common. In particular, many colorectal and endometrial tumors with MLH1/PMS2 loss have somatic MLH1 promoter methylation. This epigenetic change silences MLH1 in the tumor and usually indicates a sporadic pathway, although personal history, family history, age, constitutional epimutations, and rare exceptions may justify further assessment.
In colorectal cancer, a BRAF p.V600E finding strongly supports a sporadic MLH1-methylated pathway and is rarely associated with Lynch syndrome. BRAF testing is not used the same way in endometrial cancer. Methylation testing is more directly tied to MLH1 silencing and is interpreted by cancer type. The methylation test guide describes the difference between promoter testing and constitutional epigenetic analysis.
If abnormal tumor screening remains unexplained, germline testing can evaluate MLH1, MSH2, MSH6, PMS2, and EPCAM as appropriate. A multigene hereditary-cancer panel may be selected when the personal or family history could reflect another syndrome. Germline testing should include sequence and deletion/duplication analysis because pathogenic changes can occur through multiple variant types.
Some people have an abnormal tumor screen but no germline pathogenic variant. Paired tumor-normal sequencing may identify two acquired mismatch repair variants, explaining a “double somatic” tumor. If neither inherited nor somatic testing resolves the finding, management may depend on personal and family history rather than an assumed Lynch diagnosis.
A stable tumor does not completely exclude Lynch syndrome. False-negative MSI can occur, and some tumors associated with MSH6 or PMS2 variants may have subtle or absent instability, especially outside colorectal and endometrial cancer. Germline evaluation may still be indicated when clinical criteria are strong [2].
Treatment and Prognostic Implications
MSI-high and dMMR tumors often carry many insertion-deletion mutations and produce neoantigens that can make them visible to immune cells. Immune checkpoint inhibitors release inhibitory signals on T cells, allowing an antitumor response in some patients. MSI-high/dMMR status is therefore an important predictive biomarker across several cancers.
The exact treatment implication is not universal. It depends on whether the cancer is localized or advanced, the tumor type, line of therapy, prior treatment, resectability, and current drug approval or guideline. A pathology report may state that the biomarker can be associated with checkpoint-inhibitor benefit, but the oncologist determines whether the patient meets the relevant clinical criteria.
Not every MSI-high tumor responds, and resistance can be primary or acquired. Other molecular changes, immune microenvironment, organ site, steroid use, and disease burden may influence outcomes. Conversely, an MSS result does not mean immunotherapy can never be useful; other biomarkers and cancer-specific indications may support it.
In colorectal cancer, MSI status can also affect prognosis and adjuvant-treatment decisions. Localized MSI-high tumors often have distinct clinicopathologic features and generally favorable stage-adjusted prognosis, but stage and additional risk features remain important. Evidence about specific chemotherapy regimens should be applied through current colorectal-cancer guidance rather than generalized from the biomarker alone.
For endometrial and other cancers, MMR status is increasingly integrated into molecular classification and treatment planning. The clinical vocabulary may include dMMR, MSI-high, molecular subtype, and hereditary screening in the same report. These are related uses but should remain conceptually separate.
A biomarker detected on a small biopsy is usually considered representative enough for treatment decisions when the test is valid, but heterogeneity can occur. If a result conflicts with prior testing or clinical behavior, review of the specimen and an orthogonal method may be appropriate.
Discordant, Negative, and Limited Results
Discordance means MSI testing and immunohistochemistry do not agree. Examples include MSI-high with retained proteins or MSS with loss of one or more proteins. Causes include technical error, low tumor purity, marker limitations, unusual tumor biology, retained antigenic protein despite loss of function, treatment effects, or sample mix-up.
The response should be systematic:
- Confirm that both tests used the same tumor or understand specimen differences.
- Re-review morphology, tumor content, internal IHC controls, and staining pattern.
- Repeat the uncertain assay on another block when possible.
- Use an orthogonal method, such as PCR after IHC or NGS after PCR.
- Consider tumor sequencing, methylation, and germline evaluation based on the pattern.
A negative result can be limited by low tumor content. Normal DNA overwhelms unstable tumor alleles and can produce an MSS call. Treated tumors with few viable cells and mucinous specimens can be challenging. The laboratory’s minimum tumor requirement and quality-control statement should be checked.
Tumor type matters because MSI patterns vary. Assays initially optimized in colorectal cancer may require specific validation elsewhere. A mononucleotide PCR panel can perform well broadly, but no marker set is perfect. NGS performance also depends on the number and distribution of evaluable loci.
Immunohistochemistry can be affected by fixation, cautery, edge artifact, weak internal controls, and neoadjuvant treatment. Complete absence in tumor nuclei with retained internal control is more convincing than faint or patchy staining. Some pathogenic missense variants produce a stable but nonfunctional protein, giving retained staining despite MSI.
An indeterminate result is not equivalent to MSS or pMMR. When the biomarker could change treatment or hereditary evaluation, another specimen or method should be pursued if feasible. A 2024 review emphasizes that modern strategies include complementary molecular and protein-based approaches, especially in atypical or discordant cases [3].
Next Steps After Testing
For MSI-high or dMMR cancer, the immediate oncology question is whether the biomarker changes treatment. The hereditary question is why the repair system failed. These questions can proceed in parallel but require different follow-up.
When MLH1/PMS2 are lost, ask whether MLH1 promoter methylation was performed and whether cancer-specific BRAF testing is appropriate. When MSH2/MSH6, isolated MSH6, or isolated PMS2 loss is found, referral for germline evaluation is commonly appropriate. Age and family history can strengthen concern but should not be used to dismiss an abnormal tumor result.
When germline testing is negative, paired tumor-normal sequencing may look for two somatic hits. This can prevent labeling a family with Lynch syndrome when the tumor arose through an acquired pathway. If testing remains unresolved, genetics professionals may recommend surveillance based on the individual and family history.
Relatives should not undergo predictive testing based solely on an MSI-high tumor. Cascade testing becomes specific and informative after a germline pathogenic variant is identified. If Lynch syndrome is confirmed, relatives can be tested for that familial variant and those who carry it can follow gene-specific surveillance.
For an MSS or pMMR result, ask whether the assay was technically valid and whether hereditary testing is still indicated by the history. A normal tumor screen lowers the likelihood of Lynch syndrome in many contexts but does not eliminate all hereditary cancer risk. Other syndromes may require a broader multigene panel.
Keep the complete pathology and molecular reports. Future care may depend on the exact assay, protein pattern, tumor block, methylation result, somatic variants, and germline findings. The most accurate conclusion is not simply “MSI positive” or “MSI negative,” but a coordinated explanation of repair status, treatment relevance, and inherited-risk implications.
Discordant results deserve deliberate review. A tumor may be microsatellite stable while mismatch-repair immunohistochemistry shows loss of a protein, or it may be MSI-high while all four proteins appear retained. Low tumor content, fixation problems, limited marker performance, unusual missense variants, treatment effects, and differences among testing platforms can contribute. The appropriate response may include pathology review, repeat testing on another tumor block, an orthogonal molecular assay, tumor sequencing, or germline evaluation. Neither MSI nor immunohistochemistry should be interpreted in isolation when the patient’s age, tumor type, personal history, or family history strongly suggests Lynch syndrome. A genetics professional can integrate these findings and explain the remaining hereditary risk.
References
- Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy. 2022. College of American Pathologists guideline.
- Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: ASCO Endorsement of College of American Pathologists Guideline. 2023. Professional guideline endorsement.
- Microsatellite instability: A 2024 update. 2024. Review article.
- Genetic/Familial High-Risk Assessment: Colorectal, Endometrial, and Gastric, Version 3.2024. 2024. Clinical guideline summary.
- Universal screening of colorectal tumors for Lynch syndrome: a survey of Canadian pathologists. 2024. Practice study.
Disclaimer
This article provides general education and cannot interpret an individual MSI, mismatch repair, tumor, or germline result. Immunotherapy and hereditary-risk decisions require the complete pathology report, cancer-specific guidance, and review by oncology, pathology, and genetics professionals. Relatives should not be tested solely because a tumor is MSI-high without appropriate germline evaluation.
