Studies indicate that as many as 1.2 million individuals in the United States carry a gene mutation associated with Lynch syndrome, and that 95% of these individuals are unaware of it. 1 High levels of microsatellite instability (MSI) can be predictive of this condition, which is associated with increased cancer risk across solid tumor types including colorectal, gastric, and endometrial cancers. As we’ve discussed in previous articles such as Microsatellite Instability (MSI) and MSI for Lynch Syndrome Screening and Guiding Immunotherapy, evaluating tumor samples for MSI is an important part of understanding the etiology and management of many cancer types.
Dysregulation in DNA mismatch repair (MMR) enzyme function can cause a distinct accumulation of errors in repetitive genetic sequences known as microsatellites.1 This may result in microsatellite instability-high (MSI-H) tumors, which are generally more sensitive to immune checkpoint inhibitor treatments than microsatellite-stable (MSS) tumors. While MMR deficiency is not always synonymous with MSI-H, it is standard to screen colorectal and endometrial cancer patients for Lynch syndrome using MMR by IHC or PCR-based MSI analysis, due to the overlap of MMR deficiency and MSI status for these tumor types.2 Thus, MSI testing is not only important for precision health (detecting those individuals who are predisposed to developing cancer), but also for precision medicine (detecting those individuals who might benefit from immunotherapy).1
“16% of MSI-H cancers will be part of a germline Lynch Syndrome, which has dramatic importance for their treatment but also screening for other cancers, as well as their family members.” – Paul Walker, Chief Medical Officer at Circulogene
In 1996, an international workshop hosted by the National Cancer Institute established a set of recommendations to identify individuals with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) who should be tested for microsatellite instability testing.3 These Bethesda Guidelines were later revised in 2002, with recommendations for the classification of a cancer tumor as either microsatellite instability–high (MSI-H), microsatellite instability–low (MSI-L), or microsatellite stable (MSS), based on the number of biomarkers found with a mutation.4 According to the Bethesda/NCI panel for the detection of MSI in colorectal cancer, tumors presenting two or more unstable markers should be defined as MSI-H, whereas if no markers or only one marker is unstable the tumors are classified as MSS or MSI-L.5
One important issue when operating under these guidelines is the choice of microsatellite markers and their sensitivity, as the original NCI five-marker microsatellite panel for the evaluation of MSI may underestimate the number of MSI-H tumors.4 Several additional mononucleotide repeat microsatellites have since been proposed as sensitive and specific markers for MSI detection in cancer.5
Today, over 20 years after the introduction of the Bethesda panel, the Applied BiosystemsTM TrueMarkTM MSI Assay incorporates new features and expanded content coverage to 13 MSI markers, 8 of which are publicly known MSI markers and 5 are proprietary to Thermo Fisher Scientific. Two short tandem repeat (STR) markers are included for sample identification, preventing sample mix-up and contamination. By increasing the number of microsatellites analyzed in a panel, the TrueMark MSI Assay can provide a more accurate assessment of MSI-H, MSI-L and MSS status. With more markers to resolve cases at the edge of microsatellite instability, the TrueMark MSI assay results in fewer missed calls due to sample type or genetic phenotype.
“The new Thermo Fisher TrueMark MSI assay has made it easier than ever testing for MSI status of cancers.” – Paul Walker, Chief Medical Officer at Circulogene
The TrueMark MSI Assay is a low-input (using as little as 2 ng DNA), single-amplification solution for the rapid identification of MSI in formalin-fixed paraffin-embedded (FFPE) samples from multiple tumor types. The TrueMark MSI Assay leverages an expanded 6-dye set to incorporate additional markers and does not require a tumor-normal match, thereby cutting in half the number of wells required for each sampleand reducing the sample handling burden. The assay is compatible with the Applied BiosystemsTM SeqStudio and 3500 Series genetic analyzers.
Immunohistochemistry (IHC), a common application of immunostaining, can be used to confirm functional MSI repair status. It is used for pre-screening samples for Lynch syndrome by staining for DNA MMR enzyme activity. Samples proficient in DNA MMR are contrasted with MMR deficient samples, which are indicative of microsatellite instability. IHC is a visual technique and is largely subjective – false negative results may occur due to fixation artefacts, while false positive staining may occur in situations where amino acid substitutions lead to loss of function with preserved immunoreactive protein expression. 6 Molecular MSI analysis using PCR and fragment analysis is a more direct method to determine whether a sample is MSS or MSI-H, as it measures changes in DNA caused by MMR protein dysfunction.
Comparison Study Design
162 FFPE samples were collected from various tumor types, with over half of the samples derived from endometrial tissue, which is notoriously difficult to evaluate.7 In a head-to-head comparison of MSI stability by molecular analysis, DNA was isolated from all 162 samples and PCR products amplified using either the TrueMark MSI Assay or an alternative, “supplier P assay”. The same 162 samples were also assessed by IHC by staining for 4 different MMR proteins, where deficiency in staining of any protein would classify a sample as MMR-deficient.
162 FFPE samples were assessed using the following 3 methods:
- Immunohistochemistry (IHC) – A qualitative technique for MSI analysis that uses antibodies for protein staining for DNA MMR enzyme activity by determining if a sample is MMR-proficient (correlates to microsatellite stable (MSS)) or MMR-deficient (correlates to high microsatellite instability (MSI-H)). 4 proteins were assessed by IHC in the comparison study.
- Supplier P Assay – Molecular examination of microsatellite DNA sequences by PCR and subsequent fragment analysis. Commercial kit analyzes 5 nearly monomorphic mononucleotide microsatellite loci.
- TrueMark MSI Assay – Molecular assay with expanded panel of 13 microsatellite markers and 2 highly variable short tandem repeat (STR) sequences that can be used to track sample identity.
Table 1 summarizes the results from all 3 methods, and Figure 2 shows the distribution of “no call” results by molecular assays that were deemed MMR proficient by IHC.
|IHC||Supplier P assay||TrueMark MSI Assay|
|104 samples||102 samples||105 samples|
|IHC||Supplier P assay||TrueMark MSI Assay|
|58 samples||43 samples||53 samples|
|IHC||Supplier P assay||TrueMark MSI Assay|
|No call||No call||No call|
|0 samples||17 samples||4 samples|
Table 1. Results detailing MMR proficiency and MSI status by assay type.
Of the 104 samples determined to be MMR proficient by IHC protein staining, high concordance was observed by both the TrueMark MSI Assay and the supplier P assay (90 of the MMR proficient samples tested MSS for each assay). Notably, 9 of the MMR proficient samples gave “no call” results with supplier P assay, while only 2 were “no call” on the TrueMark MSI Assay. The two samples which were not called in the TrueMark MSI assay were also not called in supplier P’s assay – it may be that these samples were too degraded for amplification. Nearly 10% of all discordant calls were those which were deemed MMR proficient using IHC, but called MSI-H on the molecular assays, possibly due to false-positive IHC staining. For the 58 MMR deficient samples by IHC, 41 were called MSI-H by both molecular assays.
The most striking difference in classification between the two molecular assays came from samples which could not be called. Of the 162 samples, the supplier P assay deemed 17 samples as “no call”, compared to only 4 samples using the TrueMark MSI Assay. The majority of “no call” samples for the supplier P assay were called by both the TrueMark MSI Assay and IHC. Furthermore, the 4 “no call” samples from the TrueMark MSI Assay could not be analyzed by the supplier P assay either. This further demonstrates the additional resolving power driven by the additional content on the TrueMark MSI panel, which aids in the identification for stable and unstable phenotypes.
When all research samples are considered, including “no calls” and calls made manually using Applied BiosystemsTM GeneMapperTM Software, the TrueMark MSI Assay shows markedly higher sensitivity and specificity than the supplier P assay (Table 2).
|TrueMark MSI Assay: automated calls||82.7%||92.3%|
|TrueMark MSI Assay: manual calls||82.7%||93.2%|
|Supplier P assay: manual calls||70.6%||89.4%|
Table 2. Sensitivity and specificity of the TrueMark MSI Assay.
The associated TrueMark MSI Analysis Software is designed specifically for the TrueMark MSI Assay to enable automated calling of markers and samples, offering greater ease of analysis by reducing the manual QC, analysis, and review required to determine sample status. The automated calling functions without the use of a tumor-normal match, but it is also compatible with tumor-normal pairs. The TrueMark Assay MSI Analysis Software, which resulted in nearly identical performance when compared with manual calling, is particularly convenient given that 52% of the research samples were derived from the notoriously difficult endometrial tissue.6
Overall, The TrueMark MSI Assay showed excellent concordance with both the supplier P assay as well as IHC. Both the TrueMark MSI Assay and the supplier P assay agreed with the IHC assessment approximately 79% of the time, though in several research samples it was the TrueMark MSI Assay which was able to determine MSI status concordant with IHC, while the supplier P assay was unable to make a call. The TrueMark MSI Assay demonstrated both higher sensitivity and specificity than the supplier P assay, reflecting the increased utility and robustness of utilizing 13 rather than 5 markers. With its expanded panel, simplified workflow, and powerful software, the TrueMark MSI Assay empowers scientists to quickly and accurately identify MSI-H samples.
|circulating tumor DNA (ctDNA)||Fragmented DNA in the bloodstream derived from tumors|
|DNA Mismatch Repair (MMR)||A highly conserved biological pathway that plays a key role in maintaining genomic stability, dysregulation of which is a hallmark of cancer|
|Formalin Fixed Paraffin Embedded (FFPE)||A form of preservation and preparation of biopsy specimens / tissue sections|
|Hereditary Nonpolyposis Colorectal Cancer (HNPCC)||Also known as Lynch syndrome, a condition in which the tendency to develop colorectal cancer is inherited|
|Immunohistochemistry (IHC)||A laboratory method that checks for certain antigens using antibodies to stain markers within a tissue section|
|Microsatellite||A tract of repeating DNA motifs within noncoding regions of the genome, often used for linkage analysis, and inherently unstable|
|Microsatellite Instability (MSI)||An indication of genomic instability, and a condition of genetic hypermutability resulting from impaired DNA mismatch repair (MMR)|
|Microsatellite Instability-High (MSI-H)||Changes in two or more of the five National Cancer Institute-recommended markers of microsatellite instability, indicative of a high level of genomic instability|
|Microsatellite Stable (MSS)||An indication of tumor genomic stability, when none of the five microsatellite markers of the NCI Bethesda panel have been mutated|
|short tandem repeat (STR)||Often used for human identification purposes, a microsatellite with repeated units varying in length between individuals|
Download the full technical note and learn more about how to analyze microsatellite instability, here.
Be sure to catch Paul Walker’s, “Importance of Liquid Biopsy MSI for studying tumors and immunotherapy applications” here.
Dr. Paul Walker is a graduate of the Indiana University School of Medicine and Associate Professor Emeritus at the Brody School of Medicine at East Carolina University in Greenville, NC, where he served as Director of Thoracic Oncology and Director of the Hematology/Oncology Fellowship Program. He currently is Chief Medical Officer of Circulogene.
1 Haraldsdottir, Sigurdis. (2017). Microsatellite Instability Testing Using Next-Generation Sequencing Data and Therapy Implications. JCO Precision Oncology. 1-4. 10.1200/PO.17.00189.
3 Rodriguez-Bigas M, Boland C, Hamilton, S, et al. A National Cancer Institute Workshop on Hereditary Nonpolyposis Colorectal Cancer Syndrome: meeting highlights and Bethesda guidelines. J Natl Cancer Inst. 1997;89(23):1758-62.
4 Umar A, Boland CR, Terdiman JP, et al. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst. 2004;96:261-268.
5 Baudrin L, Deleuze J, How-Kit A. Molecular and Computational Methods for the Detection of Microsatellite Instability in Cancer. Fron Oncol. 2018;8:621.
6 Dedeurwaerdere F, Clase K, Dorpe J, et al. Comparison of microsatellite instability detection by immunohistochemistry and molecular techniques in colorectal and endometrial cancer. Sci Rep 11, 12880 (2021).
7 Wang Y et al. (2017) Differences in microsatellite instability profiles between endometrioid and colorectal cancers: a potential cause for false-negative results? J Mol Diag 19(1):57–64.
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