Understanding Microsatellite Instability (MSI) Testing: From Fragment Analysis to Genomic Sequencing

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Lindsay Megenhardt, PhD., CHES
Medical Affairs

What Is Microsatellite Instabilty (MSI)?

Microsatellite instability (MSI) is a genomic characteristic, most attributed to mutations or epigenetic alterations in mismatch repair (MMR) gene regions which lead to dysfunction in DNA repair mechanisms. MSI can be observed by analyzing aberrant insertions or deletions (indels) within short-tandem repeat regions or “microsatellite” regions within the genome, which assist in the identification of the MSI high (MSI-H) biomarker within solid tumors (Pećina-Šlaus et al. 2020). There are now many assays designed to identify MSI-H tumors, primarily developed around the polymerase chain reaction (PCR) fragment length analysis technique, and more recently assays using next generation sequencing (NGS) based approaches. The following summary describes the nature of each of these two approaches and compares their utility in identification of MSI-H tumors.

MSI Testing by Polymerase Chain Reaction (MSI by PCR)

MSI testing of tumors has been performed by fluorescent multiplexed PCR fragment length analysis, also referred to as MSI by PCR, since the early 2000s. This technique remains the gold-standard method for determining MSI status of solid tumors (Yamamoto & Imai 2019). MSI by PCR is a highly reproducible technique used to measure instability within a small panel of microsatellite loci regions within the human genome. Often these loci are well characterized biomarkers associated with MMR deficiency (dMMR). Many MSI by PCR assays utilize quasi-monomorphic (less variable across populations) microsatellite sequences, which improve assay performance (Bacher et al. 2004; Suraweera et al. 2002).

MSI by PCR has been used to identify MSI high (MSI-H) tumors, a biomarker for underlying tumor MMR deficiency and potential germline MMR mutation, and predictive of response to immune checkpoint blockade therapy in solid tumors (Zhao et al. 2019). This type of analysis is performed using a method called capillary electrophoresis (CE), by which PCR amplified DNA fragments for microsatellite loci can be measured from tumors and normal tissue samples. On the basis of size and electrical charge, DNA fragments separate and comparative analysis between a patient’s tumor and matched normal DNA for each PCR amplified loci is performed. If loci allele lengths within a tumor sample differ from normal germline DNA, then the loci are deemed unstable (Bacher et al. 2004; Dudley et al. 2016).

MSI Testing by Next Generation Sequencing (MSI by NGS)

Several methods have been developed to determine MSI status using NGS technology (Yamamoto & Imai 2019). Unlike most mutations detected by NGS, MSI is a genomic signature requiring sequencing information from many regions within the genome. Generation of a genomic signature requires computational approaches to determine how many regions need to be mutated for a sample to be qualitatively associated with an MSI-H status, typically as compared to MSI by PCR.

The number of microsatellite loci analyzed by MSI by NGS tests can vary widely, from 5 to 7,000. The markers analyzed may also be of varying lengths, with mono-, di-, tri-, tetra-, or pentanucleotide repeats. In addition, the bioinformatic analyses used to determine MSI status using NGS are not standardized and can differ based on factors such as the sequencing approach, number and type of MSI regions interrogated, reference methodology, algorithms, sequencing quality requirements, and established cutoffs or statistical approaches for determining MSI-H status (Deak et al. 2021; Gilson et al. 2021; Yamamoto & Imai 2019). As a result, the agreement between MSI by NGS tests and MSI by PCR can vary depending on the specific test and tumor type.

MSI status as determined by NGS analysis is typically inferred using scoring algorithms, which can vary widely between tests (Gilson et al. 2021). Some tests do not follow current NCI guidelines, which employ established thresholds for MSI-H, MSI-L, and MSS calling (Umar et al. 2004). Additionally, some tests report an alternative or "unknown" MSI result when the MSI status cannot be determined due to technical or analytical limitations or when scores fall within an equivocal range outside of accurate MSI calling thresholds as compared to PCR. These unknown calls can be categorized as:

MSI-I (indeterminate) (Froyen et al. 2022; Latham et al. 2019; PGDx 2020; Ziegler et al. 2020)
MSI-E or MSE (equivocal) (Lin et al. 2024; Rajagopal et al. 2019; Saul et al. 2020)
MSI borderline (Kang et al. 2022); or
MSI "cannot be determined" (Foundation Medicine 2023)

In these cases, this type of result from NGS does not provide an actionable clinical interpretation. This lack of definitive categorization can hinder appropriate downstream therapeutic decisions or trial eligibility assessments. Consequently, when MSI status is ambiguous, confirmatory testing using orthogonal methods such as PCR or IHC is often necessary to resolve uncertainty and ensure accurate patient stratification (Bartley et al. 2022; Lin et al. 2024; Manning-Geist et al. 2022).

Limited data is available on the incidence of MSI “unknown” calling by NGS assays, however, studies suggest this may range from ~3.2%-8.9% of solid tumor samples (Foundation Medicine 2023; Ziegler et al. 2020) . A recent large cohort study of over 191,767 solid tumor samples, found indeterminant results in 16,607 (8.66%) (Ali-Fehmi et al. 2024).

In clinical reports, “MSI Indeterminate” does not imply a biological absence of instability, but rather a technical limitation in classifying the sample with high confidence. An unknown call arises when an assay is unable to confidently classify a sample as either MSI-H or MSS. This can occur due to:

Low tumor purity, which dilutes the signal of MSI events (Bartley et al. 2022; Itkin et al. 2025)
Low DNA input or degraded FFPE samples (Itkin et al. 2025; Manning-Geist et al. 2022)
Insufficient tumor tissue available for DNA extraction (Itkin et al. 2025)
Insufficient sequence coverage at microsatellite loci (Bartley et al. 2022)

Retesting can frequently mitigate initial NGS test failures. However, since NGS assays often require substantial tissue input for adequate DNA yield, retesting may not be feasible for certain specimens, such as biopsies, where sample availability is inherently limited (Bartley et al. 2022).

Specimen Requirements

Both PCR and NGS assays evaluating MSI in solid tumors utilize FFPE tumor specimens for analysis, however, the specific requirements for tissue volume, DNA input, and tumor purity can vary greatly depending on specific assays. PCR-based assays typically require 1–2ng of DNA input for amplification reaction, however, commercial laboratories can require 1–5 unstained FFPE slides (with 20–40% tumor purity) for analysis using this method. Comparatively, NGS sequencing assays often require more DNA input to ensure sufficient sequencing coverage and depth. DNA input requirements can vary from 10–50ng or more and may be impacted by degraded DNA from FFPE samples (Cazzato et al. 2021). Similarly, minimum tissue volume requirements may be established by clinical laboratories utilizing this analysis method and range widely in FFPE slide requirements and tumor purity criteria. In general, PCR assays require less DNA input than NGS assays, however, minimum tissue specimen requirements may be lab dependent

Technical Advantages and Limitations of MSI by PCR vs. NGS

One of the major advantages to performing NGS testing in general, as compared to single biomarker testing, is that it has the potential to evaluate multiple targets simultaneously (including MMR genes). This may be critical for many advanced cancer patients to guide treatment decision making and/or assess eligibility for clinical trials. However, some MSI-H tumors occur due to epigenetic changes rather than genetic changes in MMR genes, therefore sequencing of MMR genes by NGS may not reliably predict MMR/MSI status of a tumor and may still require use of gold standard tests such as MSI by PCR and MMR by IHC for detection of MMR deficiency (Bacher et al. 2016). As such, NGS is typically used as an alternative molecular test only recommended for MSI testing when identification of other genetic aberrations is also needed to make treatment decisions and may not be recommended for cancer types with higher prevalence of MSI-H tumors (Bartley et al. 2022; Luchini et al. 2019). Table 1 outlines additional technical advantages and limitations of using PCR verses NGS for the detection of MSI in tumors.

Technical Advantages and Limitations of Using PCR v. NGS for Detection of MSI in Tumors

Method

Advantages

Limitations

MSI by PCR

Minimal sample requirements (≤1 ng DNA)
Standardized MSI markers have high sensitivity for detection of dMMR tumors
Defined indication and outcome for follow-up
Bioinformatic analyses pipeline not required
High to medium throughput (1-96 samples)
Basic molecular skillset required

Does not utilize fully automated system
Matched normal sample required (except when QMVR method is used)
Does not identify gene mutations
No indication about the MMR genes to investigate
False positives due to MSI polymorphisms (less likely with quasimonomorphic mononucleotide panels)
Moderately stringent DNA quality requirements

MSI by NGS

Can be automated
No matched normal sample requirement (varies by assay, some do require)
Simultaneous detection of other genomic alterations including MMR gene mutations
Allows for the discovery of novel variants
Data can be used for gene discovery or research purposes
High throughput (>96 samples)

Large sample requirements (>20 ng DNA)
Highly stringent DNA quality requirements
Lack of standardization and validation (Sequencing technology, MSI algorithm, Gene Panel, etc.) for detection of dMMR tumors
Potentially undefined indication/outcomes and detection of novel variants
Bioinformatic pipeline and data storage system required
False positives due to MSI polymorphisms
Highly specialized molecular skillset required

Table 1 References: (Bacher et al. 2016; Djordjevic & Broaddus 2016; Gallon et al. 2021; Gilson et al. 2021; Jang et al. 2018; Nowak & Hornick 2016; Richman 2015; Yamamoto & Imai 2019)

Clinical Importance of Evaluating MSI Using Gold Standard Methods

MSI by PCR is the gold-standard method for determining MSI status of solid tumors because it assesses quasi-monomorphic mononucleotide microsatellite loci that are sensitive for identifying tumors with deficient mismatch repair. However, many commercial NGS assays do not evaluate these loci and may assess MSI based on uninformative and problematic sequences, potentially leading to inaccurate results. Additionally, MSI by NGS assays may not require matched normal tissue, and the reference standards may not be representative of certain patient populations, which can impact MSI testing accuracy in minority populations. Indeed, a study using the commercially available MSI test from Caris has demonstrated that these issues may be significantly impacting MSI testing in African American patients (Saul et al. 2020).

Clinical Practice Guideline Recommendations for MSI Testing

The growing appreciation of the role of MSI-H status in predicting response to immune checkpoint inhibitors has resulted in a number of clinical practice guidelines regarding MSI testing globally. Professional organizations have developed recommendations for MSI or dMMR testing for the purpose of Lynch syndrome screening and/or making decisions relative to immunotherapy treatment.

North America

The College of American Pathologists (CAP) in collaboration with the Association for Molecular Pathology (AMP), American Society of Clinical Oncology (ASCO), and patient advocacy group Fight Colorectal Cancer convened a multidisciplinary expert and advisory panel to develop evidence-based guidelines to identify the optimal clinical laboratory test to identify defects in DNA mismatch repair in patients with solid tumor malignancies who are being considered for immune checkpoint inhibitor therapy (Bartley et al. 2022). The following recommendations were made by tumor type:

For patients with colorectal cancer, MMR-IHC and/or MSI-PCR are the recommended screening methods as NGS-based tumor profiling assays tend to require significantly more tissue and biopsy samples, typically the standard specimen type for CRC, can pose a challenge for NGS assays as tissue may be limited.
For upper GI tract cancers, MMR-IHC and/or MSI-PCR are recommended over MSI by NGS due to insufficient evidence to support strong recommendation of NGS in these tumors at this time.
For endometrial cancers, MMR-IHC is recommended over MSI (PCR or NGS) due to the high prevalence of MSH6 deficiency and MSI-Low frequency in this population.
For other solid tumor malignancies, the panel does not recommend an optimal methodology for DNA mismatch repair testing due to insufficient data but notes the assay used must be adequately validated for the specific tumor type being tested.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Genetic/Familial High-Risk Assessment: Colorectal, Endometrial, and Gastric V1.2025 recommend using either IHC and/or MSI as the primary approach for pathology-lab-based universal screening of all colorectal and endometrial samples for Lynch syndrome, and that screening should also be considered for sebaceous neoplasms and the following adenocarcinomas: small bowel, ovarian, gastric, pancreas, biliary tract, brain, bladder, urothelial, and adrenocortical cancers regardless of age at diagnosis. For testing, these guidelines discuss two marker panels that can be used for MSI by PCR testing, the Promega (five mononucleotide loci BAT-25, BAT-26, NR-21, NR-24, and MONO-27 and two pentanucleotide loci used for specimen identification) and the Bethesda/NCI (Two mononucleotide loci BAT-25 and BAT-26 and three dinucleotide loci D2S123, D5S346, and D17S250) panels; also noting that dinucleotide markers may be less specific than mononucleotide markers for MSI. These guidelines state that further studies are needed to determine the sensitivity and specificity of MSI by NGS compared to MMR IHC and MSI by PCR. They also state that MSI by NGS does not require confirmation by more traditional measurement of MSI by PCR or IHC if the laboratory has validated the assay for use in the cancer in which it is being used (NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®), Genetic/Familial High-Risk Assessment: Colorectal, Endometrial, and Gastric, 2025). To view the most recent and complete version of the guidelines, go online to NCCN.org.

Europe

The European Society of Medical Oncology (ESMO) published a set of guidelines in 2019 specifically addressing determination of MSI/dMMR status in solid tumors for the purpose of determining eligibility for anti-PD-1/PD-L1 immunotherapeutic treatment. IHC was the primary recommended technique for determining dMMR status due to wide availability followed by MSI by PCR based testing for MSI status. For MSI by PCR the recommended markers include five polyA mononucleotide markers (BAT-25, BAT-26, NR-21, NR-24, and NR-27). NGS panels which detect MSI were recommended for tumors where MSI prevalence is low and additional somatic mutation testing may be helpful in identifying alternative targeted therapeutics for treatment (Luchini et al. 2019).

The ESMO Precision Medicine Working Group updated the recommendations for the use of tumor NGS in patients with advanced cancers in routine practice (Mosele et al. 2024). ESMO recommends carrying out multigene NGS in patients with advanced cancers in countries where tumor-agnostic targeted therapies are accessible. Cost-effectiveness should be assessed at the local level and the decision to implement NGS should be taken accordingly.

Asia

The Japan Society of Clinical Oncology highly recommended MSI testing for determining eligibility for PD-1/PD-L1 inhibitors using an approved companion diagnostic test and includes the FALCO MSI test consisting of markers BAT-25, BAT-26, NR-21, NR-24, and MONO-27 as the approved companion diagnostic for pembrolizumab (Mishima et al. 2023). Recently, the Japanese Society of Medical Oncology published the clinical guideline for molecular testing for colorectal cancer treatment (Bando et al. 2024). The following methods are strongly recommended when assessing for MMR deficiency: MSI testing, IHC testing, and NGS-based testing.

Global

In 2020, experts in oncology representing Europe (ESMO), the United States (ASCO), and two Asian societies (JSMO and TOS) contributed to the development of recommendations for the management of patients with MSI-H tumors (Yoshino et al. 2020). There was 100% agreement among these clinical experts that patients with advanced (unresectable or metastatic) solid tumors with a high incidence of MSI/dMMR should be tested for their MSI/MMR status. For tumors with low incidence MSI/MMR testing should be considered. These experts also recommended the testing approach for determining MSI/MMR status with IHC highly recommended and PCR and validated NGS testing recommended for testing either upfront or when IHC is equivocal or not available. There was consensus that FFPE tissue blocks are the most appropriate biospecimen for this testing. PD-1/PD-L1 inhibitors were strongly recommended for patients with MSI/MMR tumors when no other satisfactory treatment options exist (Yoshino et al. 2020).

Conclusion

Microsatellite instability (MSI) testing is a critical component of tumor characterization, particularly in the context of identifying mismatch repair deficiency and informing immunotherapy eligibility. Both PCR-based and next-generation sequencing (NGS)-based approaches offer distinct advantages and limitations, and the choice of methodology should be guided by the clinical context, available tissue, and laboratory capabilities.

MSI by PCR has long served as the established standard, offering a well-validated, sensitive, and cost-effective approach for detecting MSI, particularly in tumor types with a higher prevalence of dMMR. Its use of defined microsatellite markers and minimal tissue requirements make it especially suitable when sample availability is limited.

Conversely, MSI testing by NGS enables broader genomic profiling, offering the advantage of simultaneous analysis of MSI status and other potentially actionable alterations, which can be valuable in the management of advanced cancers. However, variability in assay design, marker selection, and bioinformatic interpretation can introduce complexity, including the potential for indeterminate results. Continued efforts to standardize NGS-based MSI detection and improve analytical concordance are ongoing and promising.

Ultimately, both PCR and NGS approaches contribute valuable insights in different clinical scenarios. A comprehensive understanding of their respective strengths and limitations allows for informed decision-making and optimal patient care. In some cases, complementary testing using orthogonal methods may enhance result confidence and ensure accurate clinical interpretation.

References

Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Genetic/Familial High-Risk Assessment: Colorectal, Endometrial, and Gastric V.1.2025. © National Comprehensive Cancer Network, Inc. 2025. All rights reserved. Accessed July 1, 2025. To view the most recent and complete version of the guideline, go online to NCCN.org.

NCCN makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way.

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Disclosures and Acknowledgements

Montse Latorre, PhD. contributed content and editing of the article drafts.

What Is Microsatellite Instabilty (MSI)? MSI Testing by Polymerase Chain Reaction (MSI by PCR) MSI Testing by Next Generation Sequencing (MSI by NGS) Specimen Requirements Technical Advantages and Limitations of MSI by PCR vs. NGS Clinical Importance of Evaluating MSI Using Gold Standard Methods Clinical Practice Guideline Recommendations for MSI Testing Conclusion

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