MSI Testing in Cancer: MSI-High, PCR, IHC and Results

A practical guide to microsatellite instability, mismatch-repair deficiency, tumour samples, test methods and the terms you may see on a laboratory report.

Key takeaways

• MSI describes a pattern of DNA changes in tumour cells; dMMR describes a problem in the system that normally repairs DNA-copying errors.
• IHC examines mismatch-repair proteins, while PCR and NGS assess microsatellite instability using different laboratory approaches.
• MSI-high, MSI-low and microsatellite stable classifications depend on the validated assay, marker panel and reporting criteria used.
• Sample quality, tumour content, fixation and previous treatment can affect results or make testing inconclusive.
• An MSI-high or dMMR result may inform treatment and hereditary-risk discussions, but it does not guarantee benefit from any treatment or confirm Lynch syndrome.

When MSI testing comes up during cancer care

You may first encounter MSI on a pathology report sent by your hospital, in a molecular test result or during a consultation with an oncologist. Abbreviations such as MSI-H, MSS, dMMR and pMMR can look like a new diagnosis. They are not cancer stages. They describe biological features of the tumour that may help the care team decide whether further testing or particular treatment discussions are relevant.

Doctors commonly consider MSI or mismatch-repair testing in colorectal and endometrial cancers. Testing may also be used in gastric cancer and other solid tumours when recommended for that tumour type or clinical situation. Possible purposes include characterising the cancer, screening for signs that could point towards Lynch syndrome and assessing whether an immunotherapy discussion is appropriate.

Testing is not automatically necessary for every cancer. Its value depends on the tumour type, stage, available tissue, previous testing and how the result would affect care. Your oncologist and pathologist can clarify why it has been requested in your case and whether the result is expected to influence treatment, genetic evaluation or both.

Microsatellites, DNA repair and what instability means

Microsatellites are short stretches of DNA in which a small sequence is repeated several times. Because these repeated sections are easy to miscopy when cells divide, their length can occasionally change. A healthy mismatch-repair system works like a proofreader, identifying and correcting many of these copying errors.

Four proteins commonly assessed in this system are MLH1, PMS2, MSH2 and MSH6. If the mismatch-repair system is deficient, described as dMMR, errors can accumulate and microsatellite sequences may become longer or shorter in tumour cells. The resulting pattern is called microsatellite instability, or MSI. A functioning system is described as proficient mismatch repair, or pMMR.^[4]

MSI and MMR are closely related but are not identical measurements. MSI testing looks for the DNA footprint left by faulty repair, whereas MMR immunohistochemistry examines whether the relevant repair proteins are present in tumour cells. This distinction helps explain why results from different methods usually agree but occasionally do not.

Understanding MSI-high, MSI-low and microsatellite stable results

An MSI-high, or MSI-H, result means instability was detected across enough tested microsatellite sites to meet the assay’s threshold. Microsatellite stable, or MSS, means the test did not detect instability that met that threshold. MSI-low, or MSI-L, indicates a limited amount of instability below the level required for MSI-H.^[2]

Classification depends on the method. In historical five-marker PCR systems, instability in two or more markers was generally classified as MSI-H, one unstable marker as MSI-L and no unstable markers as MSS. Modern PCR panels and NGS algorithms may examine different markers and apply their own validated thresholds. For that reason, a category should be read alongside the assay name and the laboratory’s interpretation rather than compared by label alone.^[2]

Many clinical pathways group MSI-L with MSS because MSI-L has not consistently carried the same implications as MSI-H. Some current tests do not report MSI-L as a separate category at all. This convention can vary by tumour type and assay, so the interpretive comment on the report matters.^[3]

Which solid tumours are commonly tested and why

Colorectal and endometrial cancers are prominent settings for MSI and MMR testing because the findings can contribute to Lynch syndrome screening and may affect prognosis or treatment discussions. Gastric cancer and a range of other solid tumours may also be tested when clinical guidelines, disease stage or consideration of a biomarker-directed treatment makes the result relevant.^[4]

The meaning of a result is not uniform across cancers. An MSI or MMR finding that has an established role in one tumour type may be less common, less extensively validated or interpreted differently in another. The care team therefore considers where the tumour started, its pathology and stage, previous therapy and the reason the test was ordered.

How MSI and MMR are tested: IHC, PCR and NGS

MMR IHC assesses nuclear expression of MLH1, PMS2, MSH2 and MSH6. Complete, unequivocal loss in tumour cells, with retained staining in non-tumour internal-control cells, supports dMMR. Weak, focal, subclonal or technically uncontrolled staining should be reported as equivocal or heterogeneous and reviewed rather than forced into a dMMR or pMMR category. The loss pattern can guide additional tumour testing and possible germline evaluation.^[4]

PCR-based MSI testing examines selected microsatellite markers, usually by comparing their lengths in tumour DNA and, for some assays, matched normal DNA. Instability at a sufficient number of markers leads to an MSI-H classification. PCR directly assesses the DNA pattern, but performance depends on the selected marker panel, tumour content and the cancer type in which the assay has been validated.^[3]

Next-generation sequencing, or NGS, can assess many DNA regions computationally. MSI may be reported as part of a broader tumour panel that also examines other genomic alterations. NGS can conserve tissue by generating several biomarker results from one workflow, but algorithms, thresholds and reporting categories differ among platforms. A broad panel is not automatically preferable if a focused test answers the clinical question adequately.

MSI/MMR methods are not universally interchangeable. Current CAP guidance, endorsed by ASCO for patients being considered for immune-checkpoint therapy, recommends MMR IHC and/or MSI-PCR for colorectal cancer; a validated MSI-NGS assay may be used only if it has shown equivalence. For gastro-oesophageal adenocarcinoma and small-bowel cancer, MMR IHC and/or MSI-PCR are preferred over MSI-NGS. For endometrial cancer, MMR IHC is preferred over MSI-PCR or MSI-NGS. In other tumour types, the assay must be validated for that specific cancer. Tumour mutational burden must not be used as a substitute for MSI/MMR testing.^[4][9]

Method	What it checks	Typical sample	Strengths	Limitations
MMR IHC	Presence or loss of MLH1, PMS2, MSH2 and MSH6 proteins in tumour cells.	Formalin-fixed, paraffin-embedded (FFPE) tumour sections on slides.	Directly indicates which protein is abnormal and can guide further tumour or hereditary testing.	Interpretation depends on good fixation and internal controls; some abnormal proteins may still show staining.
PCR-based MSI assay	Length changes at selected microsatellite markers in tumour DNA, sometimes compared with matched normal DNA.	DNA extracted from FFPE tumour; some assays also use paired normal tissue or blood.	Established criteria for MSI-H, MSI-L and MSS in several tumour types; can be run on relatively small DNA amounts.	Performance depends on tumour content, marker panel and validation in the specific cancer type.
NGS-based MSI assessment	Instability patterns across many microsatellite regions, often alongside other genomic alterations.	DNA from FFPE tumour, frequently as part of a broader solid-tumour panel.	Generates MSI status plus other biomarkers from a single workflow, which can conserve tissue.	Algorithms and cut-offs differ among platforms, and not all panels have strong validation for every tumour type.

Tumour samples and pre-analytic factors that shape MSI results

Testing is usually performed on tumour tissue from a biopsy or surgical resection. The material is commonly stored as a formalin-fixed, paraffin-embedded block, often abbreviated as FFPE, or provided as unstained sections from that block. Some PCR assays request matched non-tumour tissue or blood for comparison, while other validated PCR and NGS methods can analyse tumour tissue without a paired normal sample. Germline testing for inherited risk is a separate test.^[1]

Before testing, a pathologist checks whether the selected area contains enough viable tumour. Low tumour content can dilute an unstable signal with DNA from normal cells and may produce an uncertain or falsely stable result. Small biopsies may also need to be shared among diagnosis, IHC and several molecular tests, making careful tissue planning important.

Delayed, insufficient or prolonged fixation can affect protein staining and DNA quality. Acid decalcification used for some bone specimens can damage DNA, and previous chemotherapy or radiotherapy may leave little viable tumour. If quality is inadequate, the report may be indeterminate or recommend another block, an alternative method or, when clinically justified and safe, a new specimen.^[1]

How to read the MSI/MMR section of your lab report

On many pathology and molecular reports, the MSI/MMR findings follow a similar structure. Working through them in order can make the information easier to understand.

1. Check which specimen and method were used
   Start with the specimen and method. Confirm which tumour block or biopsy was tested and whether the laboratory used MMR IHC, PCR, NGS or a combination. A result from a small treated biopsy may carry different limitations from one obtained from a larger untreated surgical specimen.
2. Find the main MSI/MMR classification
   Next, identify the main classification. “MSI-H” indicates that the molecular assay detected high-level instability, while “MSS” means it did not. “dMMR” indicates loss or dysfunction suggested by MMR protein testing; “pMMR” usually means the assessed proteins were retained. A report reading “MSI-H/dMMR” or “MSS/pMMR” shows agreement between the corresponding findings.
3. Read quality notes and any limitations
   Then read the quality and limitations statements. Terms such as “indeterminate,” “equivocal,” “borderline” or “insufficient tumour” mean that the laboratory could not make a reliable classification; they are not interchangeable with a negative result. The report may state the tumour percentage, identify an inadequate internal IHC control or note that the assay has limited validation for a particular sample or tumour type.^[1]
4. Look at the interpretive comment in context
   Finally, review the interpretive comment. It may mention possible relevance to immunotherapy, recommend review of an unexpected IHC loss pattern or suggest genetic counselling because of possible Lynch syndrome. These comments identify questions for the care team; they do not by themselves establish treatment eligibility or an inherited diagnosis.

Why IHC, PCR and NGS results can sometimes disagree

A tumour may occasionally be reported as MSI-H with retained MMR proteins, or as dMMR without a clear MSI-H result. Possible explanations include low tumour content, borderline instability, fixation or staining problems, biological differences among tumour types, a non-functional protein that is still visible by IHC, or differences in the markers and thresholds used by molecular assays.^[5]

When findings are discordant, clinicians do not usually interpret one label in isolation. The pathologist may review the original slides, confirm that IHC controls worked, reassess tumour content and check whether the assay was validated for that setting. Testing another tumour-rich block or using a complementary method may resolve the discrepancy.

If results remain uncertain, the team weighs the laboratory findings against the tumour diagnosis, personal and family history and the clinical reason for testing. An inconclusive or apparently negative result from poor-quality tissue may not settle a strong concern about mismatch-repair deficiency or inherited risk.

How MSI/MMR findings fit into treatment discussions

MSI-H or dMMR can support consideration of immune-checkpoint therapy only in defined cancer types and treatment settings; it is not a universal treatment authorization. Eligibility depends on the current jurisdiction-specific medicine label or guideline, tumour origin, stage or resectability, prior treatment, the assay accepted for that indication and the patient’s condition. Regulatory indications change, and a U.S. FDA indication should not be presented as an Indian approval.^[6][10]

An MSI-H or dMMR result does not guarantee that immunotherapy, or any other treatment, will work. Tumours with the same biomarker can respond differently, and practical decisions must also account for potential adverse effects, other biomarkers, available alternatives and the purpose of treatment. Likewise, MSS or pMMR does not prove that immunotherapy can never be relevant; another approved indication or biomarker may apply.

The report should therefore be treated as one part of a broader clinical picture. Your oncologist can determine whether the assay is accepted for the intended decision and whether the finding changes available options. Do not start, stop or select cancer treatment based only on an MSI or MMR label.

When MSI suggests possible inherited risk such as Lynch syndrome

Lynch syndrome is an inherited condition caused by a harmful germline change affecting a mismatch-repair gene. An MSI-H or dMMR tumour, particularly in colorectal or endometrial cancer, can raise the possibility of Lynch syndrome. It does not confirm the condition because mismatch-repair deficiency can also arise from changes limited to the tumour rather than changes present throughout the body.^[2]

The pattern of protein loss, additional tumour findings, age at diagnosis and personal and family history help determine whether genetic counselling or germline testing should be considered. When MLH1 and PMS2 are lost, MLH1-promoter methylation can help identify common sporadic tumours; in colorectal cancer, BRAF V600E may also be informative, but BRAF reflex testing should not be generalized to every tumour type. Germline testing remains a separate test, generally using blood or saliva.^[11]

A genetics professional can discuss the possible results, limitations and implications for relatives before or after testing. Family members should not assume they have Lynch syndrome based solely on one person’s tumour result; their screening or testing decisions require an individual risk assessment.

Accessing MSI testing in accredited laboratories in India

MSI and MMR testing is generally coordinated through an oncologist, hospital pathology department or specialised molecular laboratory. Before a sample is sent, the team should confirm the required block or slides, whether matched normal material is needed, and whether the chosen assay is suitable for the tumour type and intended clinical decision.

Verified Oncquest offering facts

1

Current listed assay details

Oncquest's current MSI Analysis listing (OMO1027) describes PCR fragment analysis requiring an FFPE tumour block, 3–4 mL EDTA blood and clinical history. The linked sample report lists BAT-25, BAT-26, NR-21, NR-24 and NR-27 and states that MSI cannot be detected reliably below 30% tumour DNA.

Why it matters for you

The method, specimen and tumour-content limitation should be checked before tissue is sent.

2

Method-name discrepancy

One public Oncquest metadata alias describes the MSI test as Sanger sequencing, while the current test listing and linked sample report describe PCR fragment analysis. Use PCR fragment analysis when describing the current offering and confirm the method with the laboratory before testing.

Why it matters for you

The test page and sample report support PCR fragment analysis; confirm this because one metadata label conflicts.

Oncquest’s MSI Analysis (OMO1027) is currently described as PCR fragment analysis requiring an FFPE tumour block, 3–4 mL of EDTA blood and clinical history. Its linked sample report shows a five-mononucleotide panel—BAT-25, BAT-26, NR-21, NR-24 and NR-27—and states that MSI cannot be detected reliably below 30% tumour DNA. Confirm current requirements, tumour content and validation for the tumour type and intended decision before sending tissue; method preference differs for colorectal, endometrial and other cancers.^[4][8][12]

Limits, uncertainties and sensible next steps

MSI/MMR testing can be highly informative, but false-negative, false-positive, borderline and discordant findings are possible. Performance depends on tumour type, specimen quality, tumour proportion, assay design and laboratory validation. A result obtained with one method cannot always be transferred directly to another clinical setting.^[5]

If a report is inconclusive, useful next steps may include pathology review, testing another tissue block, repeating the assay or using a complementary method. A new biopsy is not automatically required; its likely value and procedural risks should be considered by the treating team.

Questions worth taking to your appointment include why the test is being ordered, which method and tissue sample will be used, whether enough tumour is available, how the result could change care, and whether genetic counselling may be appropriate. If multiple results disagree, ask which finding the multidisciplinary team considers most reliable and why.

Common questions about MSI and MMR testing

FAQs

Does every person with cancer need MSI or MMR testing?

No. Testing is commonly considered in selected tumour types and clinical settings, but its usefulness depends on current guidelines, cancer stage, available tissue and whether the result could change treatment or hereditary-risk evaluation. Ask the treating oncologist what specific question the test is intended to answer.

Can a blood or saliva test replace tumour MSI testing?

Not routinely. Tumour MSI analysis assesses instability within cancer cells, while germline testing on blood or saliva looks for an inherited genetic change. Some specialised blood-based tumour assays can report MSI, but their suitability and sensitivity depend on the platform and clinical setting.

What happens if the result is indeterminate?

An indeterminate result means the sample or assay could not provide a dependable classification. The pathologist may recommend reviewing the slides, selecting a more tumour-rich block, repeating the test or using IHC, PCR or NGS as a complementary method. Whether another biopsy is appropriate depends on its likely clinical value and safety.

How do costs and access for MSI testing vary in India?

Costs vary by laboratory, test method, whether MSI is ordered alone or within a larger NGS panel, and whether extra pathology review or paired normal testing is needed. Insurance and hospital coverage also differ. Before sending tissue, request a written estimate and ask whether sample transport, pathology review and any recommended follow-up testing are included.

What should I ask before agreeing to MSI/MMR testing?

Ask which sample and method will be used, whether the laboratory has validated the assay for your tumour type, whether enough viable tumour is present, and how the result could affect care. It is also useful to ask what will happen if the result is discordant or inconclusive and whether any finding would lead to genetic counselling.

Sources

1. EMQN best practice guidelines for analysis and reporting of microsatellite instability in solid tumours - European Journal of Human Genetics / EMQN
2. Genetics of Colorectal Cancer (PDQ®) – Microsatellite Instability Pathway - National Cancer Institute
3. Molecular and Computational Methods for the Detection of Microsatellite Instability in Cancer - Frontiers in Oncology
4. Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy - College of American Pathologists
5. Testing for deficient mismatch repair and microsatellite instability: A focused update - Der Pathologe / PubMed
6. Agnostic Cancer Therapies (PDQ®): Microsatellite Instability–High or Mismatch Repair–Deficient Solid Tumors - National Cancer Institute
7. Biomarker and Tumor Marker Tests - American Cancer Society
8. MSI Analysis (Microsatellite Stability Index) Test - Oncquest Laboratories
9. Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: ASCO Endorsement - American Society of Clinical Oncology / Journal of Clinical Oncology
10. Keytruda (pembrolizumab) U.S. Prescribing Information - U.S. Food and Drug Administration
11. Genetics of Colorectal Cancer (PDQ®) – Health Professional Version - National Cancer Institute
12. MSI Analysis (OMO1027) Sample Report - Oncquest Laboratories