Determining Microsatellite Instability: The Ideal Method

Microsatellite instability (MSI) in tumors is an approved biomarker for breakthrough immune checkpoint inhibitor therapy (1), with polymerase chain reaction (PCR) testing currently the most direct, accurate, and cost-effective measurement method. In 2017, the US FDA granted the first tissue-agnostic approval for pembrolizumab – a monoclonal antibody that blocks immune suppressing PD-1/PD-1 receptor interactions, or “immune checkpoints” – for patients with unresectable or metastatic MSI or DNA mismatch repair-deficient (dMMR) solid tumors. Moreover, December 2020 saw the European Medicines Agency (EMA) adopt a new indication for pembrolizumab as a first-line treatment for metastatic colorectal cancer based on MSI or dMMR biomarker status (2).

These approvals were based on clinical trial data demonstrating that dMMR or MSI was able to predict treatment response across 12 different solid tumor types, including colorectal cancer (3,4,5). Given the compelling therapeutic rationale for measuring dMMR and MSI and the fact that MMR deficiency has been identified in up to 20 percent of different solid tumors (3), the benefits of detecting these biomarkers accurately and affordably are clear.

There’s an indelible biological link between dMMR and MSI (6). The MMR system of proteins recognizes and repairs DNA base pair mistakes, insertion and deletion errors (indels), and DNA damage that occurs during replication and recombination. Mutations in genes encoding MMR proteins can cause DNA mismatch repair defects throughout the genome, including within microsatellites – widely distributed stretches of DNA composed of short (up to six base pairs) motifs repeated up to 50 times.

Just like gene coding sequences, microsatellites can accumulate errors from dysfunctional DNA repair, including base-base mismatches and small indels that differ from the inherited microsatellite (7). MSI – the accumulation of these errors – reflects overall tumor genetic instability, whereas indels in coding sequence microsatellites may lead to frameshift mutations. In tumors driven by dysfunctional DNA repair, genetic instability is responsible for the increased tumor mutation burden that drives immune cell infiltration into the tumor microenvironment (TME) (5,6). As a survival strategy, tumors can inhibit immune cell activation in the TME by engaging immune checkpoints. MSI and dMMR can signpost tumors that are more likely to respond to immune checkpoint inhibitor therapies (3).

The MMR system includes a number of proteins including MLH1, MSH2, MSH6, and PMS2 (8) – and dMMR is often assessed in tumor cells by the absence of immunohistochemical (IHC) staining for any one of these four major proteins (9,10). MSI is detected either by PCR amplification of tumor DNA or by next-generation sequencing (NGS) methods (8,11,12); however, neither approach is optimized for MSI detection and not all platforms use the most sensitive marker panels. NGS is also expensive and not all NGS biomarkers have been clinically validated. Concordance between IHC and MSI assessment of tumors is high (approximately 90 percent), tempting pathologists to rely on one method alone (9, 13) – but, if we only employ one method for detecting MSI and dMMR, it must be the most direct, accurate, and efficient method out there.

IHC detection of dMMR

IHC detection of dMMR is based on specific antibody recognition of MLH1, MSH2, MSH6, and PMS2 in tumor cell nuclei (10), with the absence of an IHC signal indicating dMMR. IHC protocols are simple, rapid, inexpensive, and require minimal specialized instrumentation.

A distinct advantage of IHC for dMMR detection is that it reveals the identity of mutated MMR genes by lack of IHC staining – using specific antibodies against the wild-type proteins. However, IHC staining doesn’t cover all MMR genes (12), requires a tissue sample large enough to perform four separate incubations, and may be unreliable (10). For example, tumors from patients exposed to preoperative chemotherapy or radiation therapy are more difficult to assess using IHC due to artifactual loss of MSH6 protein expression (14). 

The major caveat with IHC dMMR detection is the potential disconnect between MMR protein antigenicity and function (9,10). Some missense mutations involving only a single nucleotide can lead to nonfunctional MMR proteins that are nevertheless recognized by antibodies, resulting in a false negative result (15). In addition, MMR gene mutations can code for unstable truncated proteins that can stain with the IHC test sample before degrading in the remaining tumor tissue. This leads to false negatives in up to 10 percent of samples (5,16). On the other hand, false positives can be caused by missense mutations that lead to the loss of antibody recognition without compromising protein function (9,10). Clearly, equivocal IHC results must be verified by follow-up or tangential PCR MSI testing.

PCR detection of MSI

PCR MSI detection, which reports on the functional loss of MMR proteins, is a more direct biomarker of MMR function (10,17). Tumor DNA is extracted from a 1 ng sample shaved from a formalin-fixed, paraffin-embedded block of tumor tissue, then amplified using DNA primers that span specific marker regions (loci) in microsatellites. These PCR products (amplicons) are separated according to size by capillary electrophoresis and detected based on fluorescent primer pairs. In the analysis, MSI shows up as novel peaks of DNA fragments not present in normal tissue (see Figure 1).

Microsatellite markers play a critical role in influencing MSI detection (18) and can significantly improve test sensitivity and specificity (8,11). In 1997, the National Cancer Institute recommended a reference panel of five microsatellite markers for MSI detection; known as the Bethesda panel, it consists of two mononucleotide (BAT-25 and BAT-26) and three dinucleotide (D2S123, D5S346, and D17S250) loci (19). Since then, mononucleotide microsatellite repeat sequences have proven particularly sensitive to transcription errors – making them optimal targets for measuring MSI (7,11,20).

A panel of five consensus mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, and either NR-22 or NR-27) outperformed the NCI reference panel (11, 21). The development of a commercial multiplex assay (BAT-25, BAT-26, NR-21, NR-24, and MONO-27) and the improved performance over the NCI reference panel has made this pentaplex panel the gold standard for MSI detection (7,8,20).

The quasi-monomorphic nature of these microsatellites facilitates their analysis. Their size is highly homogeneous in human populations (11,21,22), with rare allelic variants mostly in African populations (22). This makes the comparison of tumor and matching normal DNA optional in most cases, although highly recommended for ethnically diverse populations. 

Historically, according to the proportion of unstable markers, MSI status was divided into three categories: MSI-High (MSI-H), MSI-Low (MSI-L), or MS-Stable (MSS). However, because no clinical differences were observed between MSS and MSI-L tumors, it is now recommended that tumors be classified into two categories: MSI (formerly MSI-H) and MSS (formerly MSI-L and MSS). ESMO guidelines recommend including MSI-L tumors with MSS tumors (23).

Using the pentaplex panel, a tumor is considered MSI if two or more markers are unstable (24); tumors with no instability, or one unstable marker, are classified as microsatellite stable (MSS). In cases where exactly two markers are unstable, a healthy matching tissue DNA analysis is critical to confirm or reject instability due to the possible presence of rare microsatellite polymorphisms in that individual. Because it is carried out in a single PCR, the pentaplex panel is simple to use and free of errors that arise from mixing samples. It also benefits from rapid turnaround times (two to three days) and results are highly reproducible (25,26).

Recently, alternative methods based on NGS technology to establish tumors’ MSI status have been proposed (27). These methods require advanced technical capabilities and are more expensive than traditional PCR assays. Each method evaluates different microsatellites and there are few comparative studies available to fully evaluate their performance.

The clinical utility of MSI

Accurate MSI testing has had clinical significance in oncology prior to the checkpoint inhibitor indication. In a study investigating MSI prevalence across 32 different tumor types, 24 showed evidence of MSI – most commonly in early-stage disease (3). The highest incidence of MSI was in colorectal cancer (10 to 15 percent for sporadic cases) (3,24) and in endometrial cancer (17 percent) (3). For colorectal cancer, MSI is also correlated with better outcomes, indicating it as a strong prognostic factor for patient survival (28).

MSI also plays a preliminary role in diagnosing Lynch syndrome (LS) (1) – an autosomal-dominant, multicancer disorder that accounts for 2–4 percent of all colorectal cancers and 2–5 percent of endometrial cancers in women. The underlying pathogenetic drivers of LS-related cancers are germline mutations of an MMR allele, as opposed to sporadic MSI cases due to methylation of the MLH1 gene promoter. Though germline MMR genetic mutation assessment is the definitive diagnostic marker for LS, MSI testing of patients with newly diagnosed colorectal cancer can be a convenient method to confirm whether a colorectal cancer is potentially attributable to LS – 16 percent of patients with MSI-H tumors have germline mutations in MMR genes (29,30).

Beyond diagnostics, MSI has also been proposed as a screening tool for all surgical specimens from patients newly diagnosed with colorectal or endometrial cancer. ESMO guidelines further support PCR as the gold standard method, recommending MSI testing by PCR alone or with IHC to assess dMMR in any cancer type belonging to the LS spectrum (colorectal, endometrial, small intestine, urothelial, central nervous system, or sebaceous gland) (23). Once genetic mutation assessment identifies patients with LS-related cancers, physicians may then consider whether relatives should be advised to undergo screening for LS. For individuals with LS, the lifetime risks for colorectal and endometrial cancer are 70–80 percent and 40–60 percent, respectively, compared with 2 percent for the general population (1).

Consequences of inaccurate results

Though most tumors do not exhibit MSI, accurate testing provides important guidance for oncologists. It can help physicians avoid prescribing expensive immune checkpoint therapy for patients with tumors that are unlikely to respond, rule out hereditary LS and sporadic dMMR cancers in patients with a variety of LS-related malignancies, and identify their relatives’ propensity for cancer (3). It is also an important first step in guiding patients toward novel and effective immunotherapies. A key example is the KEYNOTE-016 clinical trial investigating the effects of pembrolizumab across tumor types, which demonstrated response rates of up to 53 percent, with 64 percent of those responses lasting 12 months or longer (31). There are clear clinical benefits to MSI testing – and sufficiently compelling evidence to warrant the use of PCR as the most accurate and efficient method of detection.